Antibody-drug conjugates containing hemiasterlin derivatives

Abstract

The present invention provides a hemiasterlin derivative and an antibody-drug conjugate that, when bound to an antibody, specifically inflicts cytotoxicity on target cells while suppressing cytotoxicity to normal cells. [Solution] Formula (1-1): TIFF2023062186000175.tif28149 [wherein b represents an integer of 1 to 5, and Z represents a group having a specific structure], or a salt thereof.

Description

[Technical field]

[0001] The present invention relates to a hemiasterlin derivative and an antibody-drug conjugate thereof. [Background technology]

[0002] Hemiasterlin is a natural compound having a tripeptide structure isolated from a marine sponge, and is involved in microtubule depolymerization and mitotic arrest in cells (Non-Patent Document 1).

[0003] To date, several groups have modified the structure of hemiasterlin derivatives to find hemiasterlin derivatives that exhibit strong cytotoxicity and antimitotic activity for the treatment of diseases such as cancer (Patent Documents 1 to 5, Non-Patent Documents 2 to 5). However, it has been reported that these hemiasterlin derivatives are delivered systemically because they are not target-specific, and that they also exhibit cytotoxicity against normal cells and cause side effects (Non-Patent Document 6).

[0004] An antibody-drug conjugate is a conjugate in which an antibody and a drug are bound directly or via an appropriate linker. The antibody-drug conjugate has the characteristic of suppressing systemic exposure of the drug and enhancing its efficacy on the target cells by delivering the drug to the target cells via an antibody that binds to an antigen expressed in the target cells.

[0005] In addition, several groups have reported complexes in which a hemiasterlin derivative having a maleimide group is combined with a cysteine ​​residue of an antibody or the like to form a thiosuccinimide (Patent Documents 4, 6 to 8). It has also been reported that the thiosuccinimide site undergoes reversible dissociation and binding reactions in vivo (Non-Patent Documents 7 and 8). [Prior art documents] [Patent documents]

[0006] [Patent Document 1] International Publication No. 2004 / 026293 [Patent Document 2] WO 96 / 33211 [Patent Document 3] U.S. Patent No. 7,579,323 [Patent Document 4] International Publication No. 2014 / 144871 [Patent Document 5] International Publication No. 2003 / 082268 [Patent Document 6] International Publication No. 2015 / 095952 [Patent Document 7] International Publication No. 2015 / 095953 [Patent Document 8] International Publication No. 2014 / 057436 [Non-patent literature]

[0007] [Non-Patent Document 1] Talpir, R. et al., Tetrahedron Lett., 1994, 35, 4453-4456. [Non-Patent Document 2] Zask,A. et. al.,Bioorg.Med.Chem.Lett.,2004,14,4353-4358. [Non-Patent Document 3] Zask, A. et. al., J. Med. Chem., 2004, 47, 4774-4786. [Non-Patent Document 4] Yamashita,A. et. al.,Bioorg.Med.Chem.Lett.,2004,14,5317-5322. [Non-Patent Document 5] Nieman, JA et. al., J, Nat. Prod., 2003, 66, 183-199. [Non-Patent Document 6] Rocha-Lima,CM et. al.,Cancer,2012,118,4262-4270. [Non-Patent Document 7] Alley, SC et. al.,Bioconjugate Chem.,2008,19,759-765. [Non-Patent Document 8] Baldwin, AD et. al.,Bioconjugate Chem.,2011,22, 1946-1953. Summary of the Invention [Problem to be solved by the invention]

[0008] The objective of the present invention is to provide a hemiasterlin derivative and an antibody-drug conjugate that, by binding to an antibody, specifically causes cytotoxicity to target cells while suppressing cytotoxicity to normal cells. [Means for solving the problem]

[0009] As a result of intensive research, the present inventors have found that an antibody-drug conjugate represented by formula (2-1) or (2-2) has strong antitumor activity and suppresses cytotoxicity to normal cells, thereby completing the present invention.

[0010] That is, the present invention is as follows.

[0011] [Section 1] Formula (1-1): [ka] [In the formula, b represents an integer from 1 to 5; Z is represented by the formula (Z-1), the formula (Z-2), the formula (Z-3), the formula (Za-1), the formula (Za-2), the formula (Za-3), the formula (Za-4) or the formula (Za-5): [ka] [ka] (In the formula, n represents an integer of 0 to 4, in which, when Z is a group represented by formula (Za-4) or formula (Za-5), n represents an integer of 1 to 4; AA represents a glutamic acid residue (Glu), an aspartic acid residue (Asp), or a lysine residue (Lys). When there are a plurality of AAs, each AAs may be the same or different from each other, and the AAs are bonded to each other via an amide bond. (AA) n The N-terminal nitrogen atom of forms an amide bond with the carbonyl group (a), R aa is C 1-6 represents an alkyl group, Q is represented by formula (Qa-1), formula (Qa-2), formula (Qa-3), formula (Qa-4), formula (Qa-5), formula (Qa-6) or formula (Qa-7): [ka] (In the formula, R ab and R ac are each independently a hydrogen atom, a halogen atom, or C 1-6 Alkyl group or C 1-6 represents an alkoxy group, R ad , R ae and R af each independently represents a hydrogen atom, a halogen atom, a hydroxyl group, a cyano group, an amino group, a carboxyl group, a phenyl group, or C optionally substituted with 1 to 3 fluorine atoms 1-6 Alkyl group, C 1-6 Alkoxy group or C 1-6 represents an alkyl ester group) represents a group represented by R 1a and R 1b each independently represents a hydrogen atom or a methyl group; R 2 is -(CH2) u -COR 4 represents u represents 1 or 2; R 3 and R4 are each independently -OH or -(AB) p represents AB represents Glu, Asp, or Lys, and when there are a plurality of AB, each AB may be the same or different, and AB are bonded to each other via an amide bond; p represents an integer of 1 to 4; However, R 3 or R 4 Ga-(AB) p When n and p are the same, the sum of n and p is an integer between 1 and 5. is a group represented by the formula: A compound or a salt thereof represented by the formula:

[0012] [Section 2] Formula (1-1): [ka] [In the formula, b represents an integer from 1 to 5; Z is represented by formula (Z-1), formula (Z-2) or formula (Z-3): [ka] (In the formula, n represents an integer of 0 to 4; AA represents a glutamic acid residue (Glu), an aspartic acid residue (Asp), or a lysine residue (Lys). When there are a plurality of AAs, each AAs may be the same or different from each other, and the AAs are bonded to each other via an amide bond. (AA) n The N-terminal nitrogen atom of forms an amide bond with the carbonyl group (a), Q is an unsubstituted phenyl group or a group represented by formula (Q-1): [ka] represents a group represented by R 1a and R 1beach independently represents a hydrogen atom or a methyl group; R 2 is -(CH2) u -COR 4 represents u represents 1 or 2; R 3 and R 4 are each independently -OH or -(AB) p represents AB represents Glu, Asp, or Lys, and when there are a plurality of AB, each AB may be the same or different, and AB are bonded to each other via an amide bond; p represents an integer of 1 to 4; However, R 3 or R 4 Ga-(AB) p When n and p are the same, the sum of n and p is an integer between 1 and 5. is a group represented by the formula: Item 3. The compound or salt thereof according to item 1,

[0013] [Section 3] Item 3. The compound or salt thereof according to item 1 or 2, wherein b is 2.

[0014] [Section 4] Formula (1-2): [ka] [In the formula, h represents an integer from 1 to 5; Z' is represented by the formula (Z-4), the formula (Z-5), the formula (Za-6), the formula (Za-7), the formula (Za-8), the formula (Za-9), the formula (Za-10) or the formula (Za-11): [ka] [ka] (In the formula, n represents an integer of 0 to 4, in which, when Z is a group represented by formula (Za-6), formula (Za-7), formula (Za-8), formula (Za-9), formula (Za-10) or formula (Za-11), n ​​is an integer of 1 to 4; AA represents a glutamic acid residue (Glu), an aspartic acid residue (Asp), or a lysine residue (Lys). When there are a plurality of AAs, each AAs may be the same or different from each other, and the AAs are bonded to each other via an amide bond. (AA) n The N-terminal nitrogen atom of forms an amide bond with the carbonyl group (a), Q is represented by formula (Qa-1), formula (Qa-2), formula (Qa-3), formula (Qa-4), formula (Qa-5), formula (Qa-6) or formula (Qa-7): [ka] (In the formula, R ab and R ac are each independently a hydrogen atom, a halogen atom, or C 1-6 Alkyl group or C 1-6 represents an alkoxy group, R ad , R ae and R af each independently represents a hydrogen atom, a halogen atom, a hydroxyl group, a cyano group, an amino group, a carboxyl group, a phenyl group, or C optionally substituted with 1 to 3 fluorine atoms 1-6 Alkyl group, C 1-6 Alkoxy group or C 1-6 represents an alkyl ester group) represents a group represented by R 1a and R 1b each independently represents a hydrogen atom or a methyl group. is a group represented by the formula: A compound or a salt thereof represented by the formula:

[0015] [Section 5] Formula (1-2): [ka] [In the formula, h represents an integer from 1 to 5; Z' is represented by the formula (Z-4) or the formula (Z-5): [ka] (In the formula, n represents an integer of 0 to 4; AA represents a glutamic acid residue (Glu), an aspartic acid residue (Asp), or a lysine residue (Lys). When there are a plurality of AAs, each AAs may be the same or different from each other, and the AAs are bonded to each other via an amide bond. (AA) n The N-terminal nitrogen atom of forms an amide bond with the carbonyl group (a), Q is an unsubstituted phenyl group or a group represented by formula (Q-1): [ka] represents a group represented by R 1a and R 1b each independently represents a hydrogen atom or a methyl group. is a group represented by the formula: Item 5. The compound or a salt thereof according to item 4,

[0016] [Section 6] Item 6. The compound or salt thereof according to item 4 or 5, wherein h is 5.

[0017] [Section 7] Q is a group represented by formula (Qa-1) or formula (Qa-2), R ab and R ac are each independently a hydrogen atom, a fluorine atom, a chlorine atom, a bromine atom, or C 1-6 Alkyl group or C 1-6 is an alkoxy group, R ad , R ae and R afeach independently represents a hydrogen atom, a fluorine atom, a chlorine atom, a bromine atom, or C optionally substituted by 1 to 3 fluorine atoms 1-6 Alkyl group, C 1-6 Item 5. The compound or salt thereof according to item 1 or 4, wherein R is an alkoxy group.

[0018] [Section 8] 8. The compound or salt thereof according to any one of items 1 to 7, wherein n is an integer of 0 to 2.

[0019] [Section 9] R 3 or R 4 But -(AB) p and the sum of n and p is 1 or 2, or a salt thereof according to any one of items 1 to 3.

[0020] [Section 10] (AA) n But, formula (A-1): [ka] (In the formula, AA1 and AA2 are each independently represented by Glu, Asp, or Lys.) Item 6. The compound or salt thereof according to item 2 or 5, wherein R is a group represented by the following formula:

[0021] [Section 11] (AA) n But, formula (A-2): [ka] (In the formula, AA1 and AA2 are each independently represented by Glu, Asp, or Lys.) Item 6. The compound or salt thereof according to item 2 or 5, wherein R is a group represented by the following formula:

[0022] [Section 12] R 3 and R 4 is -OH, Item 4. The compound or salt thereof according to any one of Items 1 to 3, wherein n is an integer of 0 to 2.

[0023] [Section 13] R 3 and R 4 But -(AB) p and Item 4. The compound or salt thereof according to any one of items 1 to 3, wherein n is 0 and p is 2, or n and p are each 1.

[0024] [Section 14] The compound or a salt thereof according to any one of items 1 to 3, selected from the following compounds: [ka]

[0025] [Section 15] The compound or a salt thereof according to any one of items 4 to 6, which is the following compound: [ka]

[0026] [Section 16] Formula (2-1): [ka] [In the formula, mAb stands for antibody; q represents an integer from 1 to 8; b represents an integer from 1 to 5; Z is represented by the formula (Z-1), the formula (Z-2), the formula (Z-3), the formula (Za-1), the formula (Za-2), the formula (Za-3), the formula (Za-4) or the formula (Za-5): [ka] [ka] (In the formula, n represents an integer of 0 to 4, in which, when Z is a group represented by formula (Za-4) or formula (Za-5), n represents an integer of 1 to 4; AA represents a glutamic acid residue (Glu), an aspartic acid residue (Asp), or a lysine residue (Lys). When there are a plurality of AAs, each AAs may be the same or different from each other, and the AAs are bonded to each other via an amide bond. (AA) n The N-terminal nitrogen atom of forms an amide bond with the carbonyl group (a), R aa is C 1-6 represents an alkyl group, Q is represented by formula (Qa-1), formula (Qa-2), formula (Qa-3), formula (Qa-4), formula (Qa-5), formula (Qa-6) or formula (Qa-7): [ka] (In the formula, R ab and R ac are each independently a hydrogen atom, a halogen atom, or C 1-6 Alkyl group or C 1-6 represents an alkoxy group, R ad , R ae and R af each independently represents a hydrogen atom, a halogen atom, a hydroxyl group, a cyano group, an amino group, a carboxyl group, a phenyl group, or C optionally substituted with 1 to 3 fluorine atoms 1-6 Alkyl group, C 1-6 Alkoxy group or C 1-6 represents an alkyl ester group) represents a group represented by R 1a and R 1b each independently represents a hydrogen atom or a methyl group; R 2 is -(CH2) u -COR 4 represents u represents 1 or 2; R 3 and R 4are each independently -OH or -(AB) p represents AB represents Glu, Asp, or Lys, and when there are a plurality of AB, each AB may be the same or different, and AB are bonded to each other via an amide bond; p represents an integer of 1 to 4; However, R 3 or R 4 Ga-(AB) p When n and p are the same, the sum of n and p is an integer between 1 and 5. is a group represented by the formula: An antibody-drug conjugate or a pharma- ceutically acceptable salt thereof,

[0027] [Section 17] Formula (2-1): [ka] [In the formula, mAb stands for antibody; q represents an integer from 1 to 8; b represents an integer from 1 to 5; Z is represented by formula (Z-1), formula (Z-2) or formula (Z-3): [ka] (In the formula, n represents an integer of 0 to 4; AA represents a glutamic acid residue (Glu), an aspartic acid residue (Asp), or a lysine residue (Lys). When there are a plurality of AAs, each AAs may be the same or different from each other, and the AAs are bonded to each other via an amide bond. (AA) n The N-terminal nitrogen atom of forms an amide bond with the carbonyl group (a), Q is an unsubstituted phenyl group or a group represented by formula (Q-1): [ka] represents a group represented by R 1a and R 1b each independently represents a hydrogen atom or a methyl group; R 2 is -(CH2) u -COR 4 represents u represents 1 or 2; R 3 and R 4 are each independently -OH or -(AB) p represents AB represents Glu, Asp, or Lys, and when there are a plurality of AB, each AB may be the same or different, and AB are bonded to each other via an amide bond; p represents an integer of 1 to 4; However, R 3 or R 4 Ga-(AB) p When n and p are the same, the sum of n and p is an integer between 1 and 5. is a group represented by the formula: Item 17. The antibody-drug conjugate or a pharma- ceutical acceptable salt thereof according to Item 16,

[0028] [Section 18] Item 18. The antibody-drug conjugate or a pharma- ceutical acceptable salt thereof according to item 16 or 17, wherein b is 2.

[0029] [Section 19] Formula (2-2): [ka] [In the formula, mAb stands for antibody; q represents an integer from 1 to 8; h represents an integer from 1 to 5; Z' is represented by the formula (Z-4), the formula (Z-5), the formula (Za-6), the formula (Za-7), the formula (Za-8), the formula (Za-9), the formula (Za-10) or the formula (Za-11): [ka] [ka] (In the formula, n represents an integer of 0 to 4, in which, when Z is a group represented by formula (Za-6), formula (Za-7), formula (Za-8), formula (Za-9), formula (Za-10) or formula (Za-11), n ​​is an integer of 1 to 4; AA represents a glutamic acid residue (Glu), an aspartic acid residue (Asp), or a lysine residue (Lys). When there are a plurality of AAs, each AAs may be the same or different from each other, and the AAs are bonded to each other via an amide bond. (AA) n The N-terminal nitrogen atom of forms an amide bond with the carbonyl group (a), Q is represented by formula (Qa-1), formula (Qa-2), formula (Qa-3), formula (Qa-4), formula (Qa-5), formula (Qa-6) or formula (Qa-7): [ka] (In the formula, R ab and R ac are each independently a hydrogen atom, a halogen atom, or C 1-6 Alkyl group or C 1-6 represents an alkoxy group, R ad , R ae and R af each independently represents a hydrogen atom, a halogen atom, a hydroxyl group, a cyano group, an amino group, a carboxyl group, a phenyl group, or C optionally substituted with 1 to 3 fluorine atoms 1-6 Alkyl group, C 1-6 Alkoxy group or C 1-6 represents an alkyl ester group) represents a group represented by R 1a and R 1b each independently represents a hydrogen atom or a methyl group. is a group represented by the formula: An antibody-drug conjugate or a pharma- ceutically acceptable salt thereof,

[0030] [Section 20] Formula (2-2): [ka] [In the formula, mAb stands for antibody; q represents an integer from 1 to 8; h represents an integer from 1 to 5; Z' is represented by the formula (Z-4) or the formula (Z-5): [ka] (In the formula, n represents an integer of 0 to 4; AA represents a glutamic acid residue (Glu), an aspartic acid residue (Asp), or a lysine residue (Lys). When there are a plurality of AAs, each AAs may be the same or different from each other, and the AAs are bonded to each other via an amide bond. (AA) n The N-terminal nitrogen atom of forms an amide bond with the carbonyl group (a), Q is an unsubstituted phenyl group or a group represented by formula (Q-1): [ka] represents a group represented by R 1a and R 1b each independently represents a hydrogen atom or a methyl group. is a group represented by the formula: Item 20. The antibody-drug conjugate or a pharma- ceutical acceptable salt thereof according to Item 19, wherein the antibody-drug conjugate is

[0031] [Section 21] 21. The antibody-drug conjugate or a pharma- ceutical acceptable salt thereof according to item 19 or 20, wherein h is 5.

[0032] [Section 22] Q is a group represented by formula (Qa-1) or formula (Qa-2), R ab and R ac are each independently a hydrogen atom, a fluorine atom, a chlorine atom, a bromine atom, or C 1-6 Alkyl group or C 1-6 is an alkoxy group, R ad , R ae and R af each independently represents a hydrogen atom, a fluorine atom, a chlorine atom, a bromine atom, or C optionally substituted by 1 to 3 fluorine atoms; 1-6 Alkyl group, C 1-6 20. The antibody-drug conjugate or a pharma- ceutical acceptable salt thereof according to item 16 or 19, wherein R is an alkoxy group.

[0033] [Section 23] 23. The antibody-drug conjugate or a pharma- ceutically acceptable salt thereof according to any one of items 16 to 22, wherein n is an integer of 0 to 2.

[0034] [Section 24] R 3 or R 4 But -(AB) p and the sum of n and p is 1 or 2, or a pharma- ceutically acceptable salt thereof, according to any one of items 16 to 18.

[0035] [Section 25] (AA) n But, formula (A-1): [ka] (In the formula, AA1 and AA2 are each independently represented by Glu, Asp, or Lys.) 21. The antibody-drug conjugate or a pharma- ceutical acceptable salt thereof according to item 17 or 20, wherein

[0036] [Section 26] (AA) n But, formula (A-2): [ka] (In the formula, AA1 and AA2 are each independently represented by Glu, Asp, or Lys.) 21. The antibody-drug conjugate or a pharma- ceutical acceptable salt thereof according to item 17 or 20, wherein

[0037] [Section 27] R 3 and R 4 is -OH, 19. The antibody-drug conjugate or a pharma- ceutically acceptable salt thereof according to any one of items 16 to 18, wherein n is an integer of 0 to 2.

[0038] [Section 28] R 3 and R 4 But -(AB) p and 19. The antibody-drug conjugate or a pharma- ceutically acceptable salt thereof according to any one of items 16 to 18, wherein n is 0 and p is 2, or n and p are each 1.

[0039] [Section 29] The mAbs are brentuximab, trastuzumab, inotuzumab, gemtuzumab, glembatumumab, labetuzumab, sacituzumab, rifastuzumab, indusatuzumab, polatuzumab, pinatuzumab, coltuximab, indatuximab, milatuzumab, rovalpituzumab, anetumab, tisotumab, miabetuximab, lorvotuzumab, rituximab, depatuxizumab, denintuzumab, enfortumab, tersotuzumab, bundletuzumab, sofituzumab, borsetuzumab, mirvetuximab, naratuximab, cantuzumab, and laprituximab. , bivatuzumab, vadastuximab, rupartumab, apurutumab, abagovomab, abciximab, avituzumab, abrilumab, actoxumab, adalibumab, adecatumumab, aducanumab, afacevicumab, afelimomab, alacizumab, alemtuzumab, alirocumab, altumomab, amatuximab, anatumomab, anifrolumab, anrukinzumab, apolizumab, arcitumomab, asclinbacumab, acelizumab, atezolizumab, atinumab, atorlimumab, avelumab, azintuxizumab, bapineuzumab, basilicumab Cimab, bavituximab, bectumomab, begelomab, belimumab, benralizumab, bertilimumab, besilesomab, bevacizumab, bezlotoxumab, biciromab, bimagrumab, bimekizumab, bleselumab, blinatumomab, brontuzumab, brosozumab, bococizumab, brazikumab, briakinumab, brodalumab, brolucizumab, broticutuzumab, burosumab, cabilalizumab, camrelizumab, caplacizumab, capromab, carlumab, carotuximab, catumazomab, cedelizumab, certolizumab, cetuximab, sitat Zumab, sikisumumab, serenoliximab, clivatuzumab, codrituzumab, conatumumab, concizumab, cosfrobiximab, crenezumab, crizanlizumab, cloteduumab, dacetuzumab, daclizumab, dalotuzumab, dapirorizumab, daratumumab, dectrekumab, demcizumab, denosumab, detumomab, dezamizumab, dinutuximab, zilidamab, domagurozumab, dorlimomab, drozitumab, durigotuzumab, dupilumab, durvalumab, dusigitumab, duvortuxizumab, ecromeximab, eculizumab,Edobacomab, edrecolomab, efalizumab, efungumab, eldelumab, elezamumab, elotuzumab, ersilimomab, emactuzumab, emapalumab, emibetuzumab, emicizumab, enavatuzumab, enlimomab, enoblitzumab, enokizumab, enoticumab, encituximab, epitumomab, epratuzumab, eptinezumab, erenumab, erlizumab, ertumaxomab, etaracizumab, etrolizumab, evinacumab, evolocumab, exbivirumab, faralimomab, farletuzumab, fasinumab, felvisumab , fezakinumab, ficlatuzumab, figitumumab, filibumab, flavotumab, fretikumab, fontolizumab, foralumab, foravirumab, fremanezumab, fresolimumab, fulnevetomab, fullanumab, futuximab, galcanezumab, galiximab, ganitumab, gantenerumab, gatipotuzumab, gavilimomab, gedivumab, gevokizumab, gilvetumab, girentuximab, golimumab, guselkumab, ibalizumab, ibritumomab, icrucumab, idarucizumab, ifavotuzumab, igovomab, imalumab, im Ciromab, Imgatuzumab, Inclacumab, Inebilizumab, Infliximab, Inolimomab, Intetumumab, Ipilimumab, Iratumumab, Isatuximab, Itolizumab, Ixekizumab, Keliximab, Lacnotumab, Lampalizumab, Lanadelumab, Landgrozumab, Ralcaviximab, Lebrikizumab, Remaresomab, Lenzilumab, Lerdelimumab, Lesofabutumab, Letolizumab, Lexatumumab, Ribililumab, Rifatuzumab, Ligelizumab, Rilotomab, Lintuzumab, Lirilumab, Rodericizumab, Loxobetomab, Rovo ... Zumab, rosatuximab, lucatumumab, lurizumab, lumuletuzumab, rutikizumab, mapatumumab, margetuximab, masalimoab, matuzumab, mavrilimumab, mepolizumab, metelimumab, minitelumomab, mitumomab, modotuximab, mogamulizumab, monalizumab, morolizumab, motavizumab, moxetumomab, muromonab, nacolomab, namilumab, naptumomab, narnalumab, natalizumab, navicixizumab, nabivumab, nebacumab, necitumumab, nemolizumab, nerelimomab, nesbacumab, nimotuzumab, nivolumab,Oviltoxaximab, obinutuzumab, ocaratuzumab, ocrelizumab, odulimomab, ofatumumab, olaratumab, oleculumab, orendalizumab, olokizumab, omalizumab, onartuzumab, ontuxizumab, opicinumab, oportuzumab, oregovomab, oreticumab, olticumab, otelixizumab, otlertuzumab, oxelumab, ozanezumab, ozoralizumab, pagibaximab, palivizumab, pamrevlumab, panitumumab, panobakumab, palsatuzumab, pascolizumab, pasotuxizumab, patekrisumab , patritumab, pembrolizumab, perakizumab, pertuzumab, pexelizumab, pidilizumab, placumab, prozalizumab, ponezumab, polgabiximab, prezalumab, priliximab, plitoxaximab, pritumumab, quilizumab, racotumomab, radletumab, rafivirumab, ralpancizumab, ramucirumab, ranevetomab, ranibizumab, raxibacumab, refanezumab, regavirumab, lemtolumab, reslizumab, rilotumumab, linikumab, risankizumab, rivavazumab, lobatumumab, romot ... Sozumab, Rontalizumab, Rosumantuzumab, Rovelizumab, Rozanolixizumab, Ruplizumab, Samalizumab, Sarilumab, Satralizumab, Satumomab, Secukinumab, Celicrelumab, Seribantumab, Setoxaximab, Sevirumab, Sibrotuzumab, Sifarlimumab, Siltuximab, Simtuzumab, Siplizumab, Sirukumab, Solanezumab, Solitomab, Sontuzumab, Stamulumab, Sulesomab, Sumtabumab, Suvisumab, Subratocumab, Tabalumab, Tadocizumab, Talizumab, Tamtubetomab, Tanezumab, Taplitumomab , talexuzumab, tabolixizumab, fanolesomab, nofetumomab, pintumomab, tefibazumab, terimomab, telisozumab, tenatumomab, teneliximab, teplizumab, teprotumumab, tesidolumab, tezepelumab, tigatuzumab, tildrakizumab, timigtuzumab, timolumab, tocilizumab, tomzotuximab, toralizumab, tosatoxumab, tositumomab, tobetumab, tralokinumab, tregalizumab, tremelimumab, trevoglumab, tucotuzumab, tuvilumab, ublituximab, urocuprumab, urelumab,Item 29. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of Items 16 to 28, which is an antibody selected from the group consisting of urtoxazumab, ustekinumab, utomilumab, vanticutumab, vanucizumab, bapaliximab, valisacumab, varlilumab, batelizumab, vedolizumab, beltuzumab, beparimomab, besencumab, visilizumab, bovalilizumab, volociximab, bonlerolizumab, votumumab, bunakizumab, tacatuzumab, zaltuzumab, zanolimumab, diralimumab, zolimomab, camidanlumab, cofetuzumab, radilatuzumab, loncastuzumab, terisotuzumab, enapotamab, and AMG595, or an anti-emvidin antibody.

[0040] [Section 30] Item 30. The antibody-drug conjugate or a pharma- ceutical acceptable salt thereof according to Item 29, wherein the mAb is brentuximab, trastuzumab, inotuzumab, gemtuzumab, labetuzumab, polatuzumab, cortuximab, indatuximab, anetuzumab, rituximab, denintuzumab, laprituximab, vadastuximab, glembatumumab, cetuximab, alemtuzumab, or depatuxizumab.

[0041] [Section 31] Item 31. The antibody-drug conjugate or a pharma- ceutical acceptable salt thereof according to Item 30, wherein the mAb is brentuximab or trastuzumab.

[0042] [Section 32] A pharmaceutical composition comprising the antibody-drug conjugate or a pharma- ceutically acceptable salt thereof according to any one of items 16 to 31.

[0043] [Section 33] Item 32. The antibody-drug conjugate or a pharma- ceutical acceptable salt thereof according to any one of items 16 to 31, and one or more anticancer compounds selected from the group consisting of anticancer alkylating agents, anticancer antimetabolites, anticancer antibiotics, anticancer platinum coordination compounds, anticancer camptothecin derivatives, anticancer tyrosine kinase inhibitors, anticancer serine threonine kinase inhibitors, anticancer phospholipid kinase inhibitors, anticancer monoclonal antibodies, interferons, biological response modifiers, hormones, immune checkpoint inhibitors, epigenetics-related molecule inhibitors, and protein post-translational modification inhibitors, or pharma- ceutical compositions comprising the above-mentioned anticancer compounds and ...

[0044] [Section 34] 32. An anticancer agent comprising the antibody-drug conjugate or a pharma- ceutically acceptable salt thereof according to any one of items 16 to 31.

[0045] [Section 35] Item 35. The anticancer agent according to Item 34, wherein the cancer is breast cancer, stomach cancer, lung cancer, liver cancer, cervical cancer, colorectal cancer, rectal cancer, colon cancer, glioma, lymphoma, ovarian cancer, pancreatic cancer, prostate cancer, kidney cancer, urothelial cancer, skin cancer, thyroid cancer, bladder cancer, head and neck cancer, uterine cancer, mesothelioma, melanoma, multiple myeloma, or leukemia.

[0046] [Section 36] A method for treating cancer, comprising administering the antibody-drug conjugate or a pharma- ceutically acceptable salt thereof according to any one of items 16 to 31 to a patient in need of such treatment.

[0047] [Section 37] Item 16. Use of the compound or a salt thereof according to any one of items 1 to 15 for the production of an anticancer agent.

[0048] [Section 38] Item 32. Use of the antibody-drug conjugate or a pharma- ceutically acceptable salt thereof according to any one of Items 16 to 31 for the manufacture of an anticancer agent.

[0049] [Section 39] Item 32. The antibody-drug conjugate or a pharma- ceutically acceptable salt thereof according to any one of items 16 to 31, for use in the treatment of cancer.

[0050] [Section 40] Item 32. The antibody-drug conjugate or the pharma- ceutical acceptable salt thereof according to any one of Items 16 to 31, for use in combination with one or more anticancer compounds or pharma-ceutical acceptable salts thereof selected from the group consisting of anticancer alkylating agents, anticancer antimetabolites, anticancer antibiotics, anticancer platinum coordination compounds, anticancer camptothecin derivatives, anticancer tyrosine kinase inhibitors, anticancer serine-threonine kinase inhibitors, anticancer phospholipid kinase inhibitors, anticancer monoclonal antibodies, interferons, biological response modifiers, hormonal agents, immune checkpoint inhibitors, epigenetics-related molecule inhibitors, and protein post-translational modification inhibitors, for treating cancer. Effect of the Invention

[0051] The antibody-drug conjugate formed between the hemiasterlin derivative of the present invention and an antibody exhibits specific cytotoxic activity against antigen-expressing cells and has low cytotoxicity in normal cells other than antigen-expressing cells, and therefore can be used as an anticancer drug with excellent safety. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0052] In this specification, "C 1-6 The term "alkyl group" means a linear or branched saturated hydrocarbon group having 1 to 6 carbon atoms. 1-6 As the alkyl group, preferably "C 1-4 More preferably, "C 1-3 Preferred examples include an alkyl group, a methyl group, an ethyl group, a propyl group, or an isopropyl group, and particularly preferred examples include a methyl group or an ethyl group.

[0053] "C 1-3 Specific examples of the alkyl group include a methyl group, an ethyl group, a propyl group, and an isopropyl group. 1-4 Specific examples of the alkyl group include, for example, "C 1-3In addition to the specific examples of the alkyl group, examples of the alkyl group include a butyl group, a 1,1-dimethylethyl group, a 1-methylpropyl group, and a 2-methylpropyl group. 1-6 Specific examples of the alkyl group include "C 1-4 In addition to those given as specific examples of "alkyl group," examples include a pentyl group, a 3-methylbutyl group, a 2-methylbutyl group, a 2,2-dimethylpropyl group, a 1-ethylpropyl group, a 1,1-dimethylpropyl group, a hexyl group, a 4-methylpentyl group, a 3-methylpentyl group, a 2-methylpentyl group, a 1-methylpentyl group, a 3,3-dimethylbutyl group, a 2,2-dimethylbutyl group, a 1,1-dimethylbutyl group, and a 1,2-dimethylbutyl group.

[0054] In this specification, "C 1-6 An "alkyl ester" is an ester (-COOR') in which R' is the above-mentioned "C 1-6 "C" refers to an ester that is an "alkyl group." 1-6 As the "alkyl ester", preferably "C 1-4 More preferably, "C 1-3 Preferred examples include methyl ester, ethyl ester, isopropyl ester, and tertiary butyl ester, and particularly preferred examples include methyl ester and ethyl ester.

[0055] "C 1-3 Specific examples of "alkyl ester" include, for example, methyl ester, ethyl ester, propyl ester, and isopropyl ester. 1-4 Specific examples of "alkyl esters" include "C 1-3 In addition to the specific examples of "alkyl esters," butyl esters, tertiary butyl esters, etc. are also included. 1-6 Specific examples of "alkyl esters" include "C 1-4In addition to the specific examples of "alkyl ester," examples thereof include pentyl ester, 3-methylbutyl ester, 2-methylbutyl ester, 2,2-dimethylpropyl ester, 1-ethylpropyl ester, 1,1-dimethylpropyl ester, hexyl ester, 4-methylpentyl ester, 3-methylpentyl ester, 2-methylpentyl ester, 1-methylpentyl ester, 3,3-dimethylbutyl ester, 2,2-dimethylbutyl ester, 1,1-dimethylbutyl ester, and 1,2-dimethylbutyl ester.

[0056] In this specification, "C 1-6 An alkoxy group is a 1-6 "C" means an oxy group substituted with an alkyl group. 1-6 As the "alkoxy group", preferably "C 1-4 An alkoxy group is preferably used. 1-3 Preferred examples of the alkoxy group include a methoxy group, an ethoxy group, a propoxy group, or a 1-methylethoxy group, and particularly preferred examples include a methoxy group or an ethoxy group.

[0057] "C 1-3 Specific examples of the "alkoxy group" include a methoxy group, an ethoxy group, a propoxy group, and a 1-methylethoxy group. 1-4 Specific examples of the "alkoxy group" include "C 1-3 In addition to the specific examples of the "alkoxy group," examples of the "alkoxy group" include a butoxy group, a 1,1-dimethylethoxy group, a 1-methylpropoxy group, and a 2-methylpropoxy group. 1-6 Specific examples of the "alkoxy group" include "C 1-4In addition to the examples given as specific examples of the "alkoxy group," examples include a pentyloxy group, a 3-methylbutoxy group, a 2-methylbutoxy group, a 2,2-dimethylpropoxy group, a 1-ethylpropoxy group, a 1,1-dimethylpropoxy group, a hexyloxy group, a 4-methylpentyloxy group, a 3-methylpentyloxy group, a 2-methylpentyloxy group, a 1-methylpentyloxy group, a 3,3-dimethylbutoxy group, a 2,2-dimethylbutoxy group, a 1,1-dimethylbutoxy group, and a 1,2-dimethylbutoxy group.

[0058] In the present specification, the term "halogen atom" includes a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom. Preferably, the halogen atom is a fluorine atom or a chlorine atom, and more preferably, the halogen atom is a fluorine atom.

[0059] <Hemiastarin derivatives> The compound represented by formula (1-1) or formula (1-2) and a salt thereof (hereinafter sometimes referred to as the "hemiasterlin derivative of the present invention") are as follows.

[0060] (1) Compounds represented by formula (1-1) and salts thereof First, among the hemiasterlin derivatives of the present invention, the compound represented by the following formula (1-1) and a salt thereof will be described. [ka]

[0061] In the formula, b represents an integer of 1 to 5. That is, b is 1, 2, 3, 4, or 5. One embodiment of b is an integer of 1 to 4, another embodiment is an integer of 1 to 3, and another embodiment is 2 or 3.

[0062] In the formula, Z represents the formula (Z-1), the formula (Z-2), the formula (Z-3), the formula (Za-1), the formula (Za-2), the formula (Za-3), the formula (Za-4), the formula (Za-5): [ka] [ka] It represents a group represented by the following formula:

[0063] In these formulas, AA represents a glutamic acid residue, an aspartic acid residue, or a lysine residue, preferably a glutamic acid residue or an aspartic acid residue. In this specification, unless otherwise required, the following three-letter abbreviations may be used to represent both α-amino acids and the corresponding amino acid residues. In addition, the optical activity of α-amino acids may include any of DL-, D-, or L-isomers unless otherwise specified. For example, "glutamic acid" or "Glu" represents DL-glutamic acid or a residue thereof, D-glutamic acid or a residue thereof, or L-glutamic acid or a residue thereof. Asp…aspartic acid, Glu…glutamic acid, Lys…lysine

[0064] In these formulas, n represents an integer of 0 to 4. That is, n is 0, 1, 2, 3, or 4. One embodiment of n is an integer of 0 to 2, another embodiment is 0 or 1, another embodiment is 1 or 2, another embodiment is an integer of 1 to 3, and another embodiment is 0. However, when Z is a group represented by formula (Za-4) or formula (Za-5), n is an integer of 1 to 4.

[0065] When there are multiple AAs, each AAs may be the same or different, and the AAs are bonded to each other via an amide bond. "AAs are bonded to each other via an amide bond" means that the carboxyl group of one amino acid and the amino group of another amino acid are condensed to form an amide bond. For example, when n is 2 and both AAs are Glu, the nitrogen atom (d) of one Glu and the carbonyl group (c) of the other Glu may be linked to each other by forming an amide bond as shown in the following formula. [ka]

[0066] (AA) n The N-terminal nitrogen atom of AA forms an amide bond together with the carbonyl group (a). For example, when AA is Asp, the nitrogen atom (b) and the carbonyl group (a) of Asp are linked together by forming an amide bond as shown in the following formula: [ka]

[0067] (AA) n An embodiment of the formula (A-1) or (A-2) in which n is 2 is given. [ka] In these formulas, AA1 and AA2 are each independently represented by Glu, Asp, or Lys.

[0068] In the formulas (Z-1), (Z-2), and (Z-3), Q is a formula (Qa-1), (Qa-2), (Qa-3), (Qa-4), (Qa-5), (Qa-6), or (Qa-7): [ka] It represents a group represented by the following formula:

[0069] In formula (Qa-1), R ab and R ac are each independently a hydrogen atom, a halogen atom, or C 1-6 Alkyl group or C 1-6 In formula (Qa-2), R represents an alkoxy group. ad , R ae and R af each independently represents a hydrogen atom, a halogen atom, a hydroxyl group, a cyano group, an amino group, a carboxyl group, a phenyl group, or C optionally substituted with 1 to 3 fluorine atoms1-6 Alkyl group, C 1-6 Alkoxy group or C 1-6 Represents an alkyl ester group. The halogen atom may be, for example, a fluorine atom, a chlorine atom or a bromine atom.

[0070] Q may be a group represented by formula (Qa-2) or formula (Qa-1), and is an unsubstituted phenyl group or a group represented by formula (Q-1): [ka] The group may be represented by the following formula:

[0071] In formula (Z-1), formula (Z-2), formula (Z-3), formula (Za-4), and formula (Za-5), R 1a and R 1b R each independently represents a hydrogen atom or a methyl group. 1a is a methyl group, and R 1b is preferably a hydrogen atom.

[0072] In this specification, a hydrogen atom is 1 Even if it is H. 2 H(D). That is, for example, one or more compounds represented by formula (1-1) 1 H 2 Deuterium converted to H(D) is also included in the compound represented by formula (1-1).

[0073] In formula (Z-1) and formula (Za-1), R 2 Ha-(CH2) u -COR 4 where u is 1 or 2, and R 4 is -OH or -(AB) pAB represents Glu, Asp, or Lys, and when there are a plurality of AB, each AB may be the same or different, and AB are bonded to each other via an amide bond. AB is preferably Glu or Asp. p is an integer of 1 to 4, that is, 1, 2, 3, or 4. One embodiment of p is 1 or 2, another embodiment is 1, another embodiment is 2, another embodiment is an integer of 1 to 3, and another embodiment is 3.

[0074] In formula (Z-2), formula (Z-3), formula (Za-2) and formula (Za-3), R 3 is -OH or -(AB) p Here, -(AB) p has the same meaning as that in formula (Z-1) and formula (Za-1).

[0075] In formula (Z-1), formula (Z-2), formula (Z-3), formula (Za-1), formula (Za-2) and formula (Za-3), R 3 or R 4 Ga-(AB) p In the above formula, the sum of n and p is an integer of 1 to 5, i.e., 1, 2, 3, 4, or 5. One embodiment of the sum of n and p is an integer of 1 to 4, another embodiment is an integer of 1 to 3, and another embodiment is 1 or 2.

[0076] In formula (Za-1), formula (Za-2) and formula (Za-3), R aa is C 1-6 R is an alkyl group. aa may be, for example, a methyl group, an ethyl group, a propyl group, an isopropyl group, and the like.

[0077] In one embodiment, Z may be a group represented by formula (Z-1), formula (Z-2) or formula (Z-3), and Q may be an unsubstituted phenyl group or a group represented by formula (Q-1).

[0078] In formula (1-1), R 3 , R 4As an embodiment of the combination of and n, R 3 and R 4 represents -OH, and n is an integer of 0 to 2. 3 , R 4 As an embodiment of the combination of n and p, R 3 and R 4 Ga-(AB) p wherein n is 0 and p is 2, or n and p are 1.

[0079] One embodiment of the hemiasterlin derivative of the present invention is the following (1-1-A). (1-1-A) In formula (1-1), b is 2, 3, 4 or 5; n is an integer of 0 or 1; Z is a group represented by formula (Z-1), formula (Z-2) or formula (Z-3), Q is an unsubstituted phenyl group or a group represented by formula (Q-1), R 1a is a methyl group, R 1b is a hydrogen atom, AA is Glu or Asp; R 2 But -(CH2) u -COR 4 and u is an integer of 1 or 2; R 3 is -OH or -(AB) p and R 4 is -OH or -(AB) p and p is an integer of 1 or 2; AB represents Glu or Asp, and when there are a plurality of AB, each AB may be the same or different, and AB are bonded to each other via an amide bond; R 3 or R 4 Ga-(AB) p when the sum of n and p is 1 or 2, A compound or a salt thereof.

[0080] One embodiment of the hemiasterlin derivative of the present invention includes any one of the following compounds or a salt thereof. [ka]

[0081] (2) Compounds represented by formula (1-2) and salts thereof Next, among the hemiasterlin derivatives of the present invention, the compound represented by the following formula (1-2) and a salt thereof will be described. [ka]

[0082] In the formula, h represents an integer of 1 to 5. That is, h is 1, 2, 3, 4, or 5. One embodiment of h is an integer of 2 to 5, another embodiment is an integer of 3 to 5, and another embodiment is 4 or 5.

[0083] In the formula, Z' is a formula (Z-4), a formula (Z-5), a formula (Za-6), a formula (Za-7), a formula (Za-8), a formula (Za-9), a formula (Za-10), or a formula (Za-11): [ka] [ka] where n, AA, Q, and R 1a and R 1b are the same as those in formula (1-1), provided that when Z' is a group represented by formula (Za-6), formula (Za-7), formula (Za-8), formula (Za-9), formula (Za-10) or formula (Za-11), n ​​is an integer of 1 to 4.

[0084] In one embodiment, Z' may be a group represented by formula (Z-4) or formula (Z-5), and Q may be an unsubstituted phenyl group or a group represented by formula (Q-1).

[0085] One embodiment of the hemiasterlin derivative of the present invention is the following (1-2-A). (1-2-A) In formula (1-2), h is 2, 3, 4 or 5; Z' is a group represented by formula (Z-4) or formula (Z-5), Q is an unsubstituted phenyl group or a group represented by formula (Q-1), R 1a is a methyl group, R 1b is a hydrogen atom, AA is Glu or Asp; n is 0, 1, or 2; A compound or a salt thereof.

[0086] One embodiment of the hemiasterlin derivative of the present invention includes the following compound or a salt thereof. [ka]

[0087] <Antibody-drug conjugates> The antibody-drug conjugate represented by formula (2-1) or formula (2-2) or a pharma- ceutically acceptable salt thereof (hereinafter sometimes referred to as the "antibody-drug conjugate of the present invention") is a conjugate in which an antibody moiety derived from an antibody molecule and a drug moiety derived from a drug molecule are directly covalently bonded, as shown below. In this specification, the "antibody-drug conjugate" is also referred to as an "ADC". [ka]

[0088] In formulae (2-1) and (2-2), Z, Z', b and h have the same meanings as those in formulae (1-1) and (1-2).

[0089] q indicates the drug antibody ratio (also referred to as Drug Antibody Ratio, or DAR) in the antibody-drug conjugate molecules represented by formula (2-1) and formula (2-2). The drug antibody ratio q means the number of drug molecules per antibody molecule in one antibody-drug conjugate molecule, i.e., per antibody-drug conjugate molecule. Note that an antibody-drug conjugate obtained by chemical synthesis is often a mixture of multiple antibody-drug conjugate molecules that may have different drug antibody ratios q. In this specification, the overall drug antibody ratio in such a mixture of antibody-drug conjugates (i.e., the average value of the drug antibody ratios q of each antibody-drug conjugate) is referred to as the "average drug antibody ratio" or "average DAR".

[0090] q is 1, 2, 3, 4, 5, 6, 7, or 8. In one embodiment, q is an integer from 2 to 8, in another embodiment, an integer from 2 to 6, in another embodiment, an integer from 4 to 8, in another embodiment, an integer from 6 to 8, in another embodiment, 2 or 4, in another embodiment, 6 or 8, and in another embodiment, 8.

[0091] One embodiment of the average DAR is 2 to 8, another embodiment is 3.5 to 4.5, and another embodiment is 1 to 2, 2 to 3, 3 to 4, 4 to 5, 5 to 6, 6 to 7, or 7 to 8. The average DAR can be determined by a method commonly used for determining the average DAR, such as SDS-PAGE, mass spectrometry, ELISA (enzyme-linked immunosorbent assay), HPLC (high performance liquid chromatography), etc. An antibody-drug conjugate having a specific DAR can be separated, purified, and characterized from a mixture of antibody-drug conjugates having different DARs by a method such as hydrophobic interaction column chromatography (HIC) HPLC, reversed-phase HPLC, electrophoresis, etc.

[0092] In formula (2-1) and formula (2-2), the antibody mAb is not particularly limited as long as it is an antibody capable of recognizing an antigen present on the surface of a target cell. The target cell may be any cell that requires treatment with a hemiasterlin derivative, and is preferably a cancer cell. The antigen present on the surface of the target cell is preferably an antigen specific to the target cell, which is not expressed or is expressed in a small amount on normal cells. In one embodiment, the mAb may be any of the known antibodies listed above. In another embodiment, the mAb may be any of brentuximab, trastuzumab, inotuzumab, gemtuzumab, glembatumumab, labetuzumab, sacituzumab, rifastuzumab, indusatuzumab, polatuzumab, pinatuzumab, cortuximab, indatuximab, milatuzumab, rovalpituzumab, anetuzumab, tisotumab, miabetuximab, lorvotuzumab, rituximab, depatuximab, and dentin. Tuzumab, enfortumab, tersotuzumab, bundletuzumab, sofituzumab, borsetuzumab, mirvetuximab, naratuximab, cantuzumab, raprituximab, bivatuzumab, vadastuximab, rupartumab, apurutuzumab, abagovomab, abciximab, avituzumab, abrilumab, actoxumab, adalibumab, adecatumumab, aducanumab, afacevicumab, afelimomab, alacizumab, alemtuzumab, alirocumab , altumomab, amatuximab, anatumomab, anifrolumab, anrukinzumab, apolizumab, arcitumomab, asclinbacumab, acelizumab, atezolizumab, atinumab, atorolimumab, avelumab, azintuximab, bapinezumab, basiliximab, bavituximab, bectumomab, begelomab, belimumab, benralizumab, bertilimumab, besilesomab, bevacizumab, bezlotoxumab, biciromab, bimagrumab, bimeki Zumab, bleselumab, blinatumomab, brontuzumab, brosozumab, bococizumab, brazikumab, briakinumab, brodalumab, brolucizumab, brotictuzumab, burosumab, cabilalizumab, camrelizumab, caplacizumab, capromab, carlumab, carotuximab, catumazomab, cedelizumab, certolizumab, cetuximab, sitatuzumab, sikisumab, serenoliximab, clivatuzumab, codrituzumab, conatumumab,Concizumab, cosfrobiximab, crenezumab, crizanlizumab, cloteduumab, dacetuzumab, daclizumab, darotuzumab, dapirorizumab, daratumumab, dectrekumab, demcizumab, denosumab, detumomab, desamizumab, dinutuximab, zilidabumab, domagrozumab, dorlimomab, drozitumab, durigotuzumab, dupilumab, durvalumab, dusigitumab, duvortuxizumab, ecloneximab, eculizumab, edovacomab, edrecolomab, efalizumab, efungumab, eldelumab, elezamumab ... , elotuzumab, ersilimomab, emactuzumab, emapalumab, emibetuzumab, emicizumab, enavatuzumab, enlimomab, enoblitzumab, enokizumab, enoticumab, encituximab, epitumomab, epratuzumab, eptinezumab, erenumab, erlizumab, ertumaxomab, etaracizumab, etrolizumab, evinacumab, evolocumab, exbivirumab, faralimomab, farletuzumab, fasinumab, felvizumab, fezakinumab, ficlatuzumab, figitumumab, filibumab, flavotumab, Fretikumab, fontolizumab, foralumab, foravirumab, fremanezumab, fresolimumab, furunevetomab, furanumab, futuximab, galcanezumab, galiximab, ganitumab, gantenerumab, gatipotuzumab, gavilimomab, gedivumab, gevokizumab, gilvetomab, girentuximab, golimumab, guselkumab, ibalizumab, ibritumomab, icrucumab, idarucizumab, ifavotuzumab, igovomab, imalumab, imciromab, imgatuzumab, inlacumab, inebilizumab, infliximab, ino Rimomab, Intetumumab, Ipilimumab, Iratumumab, Isatuximab, Itolizumab, Ixekizumab, Keliximab, Lacnotumab, Lampalizumab, Lanadelumab, Landgrozumab, Ralcaviximab, Lebrikizumab, Remaresomab, Lenzilumab, Lerdelimumab, Lesofabutumab, Letolizumab, Lexatumumab, Ribililumab, Rifatuzumab, Ligelizumab, Rilotomab, Lintuzumab, Lirilumab, Roderucizumab, Lokivetomab, Rolovotuzumab, Rosatuximab, Lucatumumab, Lulizumab, Lumuletuzumab, Rutikizumab,Mapatumumab, margetuximab, masalimoab, matuzumab, mavrilimumab, mepolizumab, metelimumab, minitelumomab, mitumomab, modotuximab, mogamulizumab, monalizumab, morolizumab, motavizumab, moxetumomab, muromonab, nacolomab, namilumab, naptumomab, narnalumab, natalizumab, navicixizumab, nabivumab, nebacumab, necitumumab, nemolizumab, nerelimomab, nesbacumab, nimotuzumab, nivolumab, obiltoxaximab, obinutuzumab, ocaratuzumab, ocrelizumab, oduli Momab, ofatumumab, olaratumab, oleculab, orendalizumab, olokizumab, omalizumab, onartuzumab, ontuxizumab, opicinumab, oportuzumab, oregovomab, oreticumab, olticumab, otelixizumab, otlertuzumab, oxelumab, ozanezumab, ozoralizumab, pagibaximab, palivizumab, pamrevlumab, panitumumab, panobacumab, palsatuzumab, pascolizumab, pasotuxizumab, pateclizumab, patritumab, pembrolizumab, perakizumab, pertuzumab, pexelizumab, Pidilizumab, placulumab, prozalizumab, ponezumab, polgabiximab, prezalumab, priliximab, plitoxaximab, pritumumab, quilizumab, racotumomab, radletumab, rafivirumab, ralpancizumab, ramucirumab, ranevetomab, ranibizumab, raxibacumab, refanezumab, regavirumab, lemtolumab, reslizumab, rilotumumab, linikumab, risankizumab, rivavazumab, lobatumumab, loredumab, romosozumab, rontalizumab, rosmantuzumab, rovelizumab, rozanolixizumab, ru Prizumab, Samalizumab, Sarilumab, Satralizumab, Satumomab, Secukinumab, Celicrelumab, Seribantumab, Setoxaximab, Sevirumab, Sibrotuzumab, Sifalimumab, Siltuximab, Simtuzumab, Siplizumab, Silkumab, Solanezumab, Solitomab, Sontuzumab, Stamulumab, Sulesomab, Sumtabumab, Suvisumab, Subratocumab, Tabalumab, Tadocizumab, Talizumab, Tamtubetomab, Tanezumab, Taplitumomab, Talexuzumab, Tabolixizumab, Fanolesomab, Nofetumomab, Pintumomab,Tefibazumab, terimomab, telisozumab, tenatumomab, teneliximab, teplizumab, teprotumumab, tesidolumab, tezepelumab, tigatuzumab, tildrakizumab, timigtuzumab, timolumab, tocilizumab, tomzotuximab, toralizumab, tosatoxumab, tositumomab, tobetumab, tralokinumab, tregalizumab, tremelim Mab, trevoglumab, tucotuzumab, tuvilumab, ublituximab, urocupulumab, urelumab, urtoxazumab, ustekinumab, utomilumab, vanticutumab, vanucizumab, bapaliximab, valisacumab, varlilumab, batelizumab, vedolizumab, beltuzumab, bepalimomab, besencumab, visilizumab, bovalilizumab, boroxicam Examples of the anti-embidiin antibody include simab, bonlerolizumab, votumumab, bunakizumab, tacatuzumab, zaltuzumab, zanolimumab, diralimumab, zolimomab, and anti-embidiin antibody. In another embodiment, brentuximab, trastuzumab, inotuzumab, gemtuzumab, labetuzumab, polatuzumab, cortuximab, indatuximab, anetumab, rituximab, denizumab, and the like. Examples of the antibody include entuzumab, laprituximab, vadastuximab, glembatumumab, cetuximab, alemtuzumab, and depatuximab. Another embodiment includes brentuximab, trastuzumab, rituximab, or an anti-emvidin antibody. Another embodiment includes brentuximab or trastuzumab, and preferably brentuximab.

[0093] Another embodiment of the mAb includes anti-19A antibody, anti-AXL antibody, anti-BCMA antibody, anti-C4.4a antibody, anti-CA6 antibody, anti-CA9 antibody, anti-CA-125 antibody, anti-cadherin-6 antibody, anti-CD166 antibody, anti-CD 19 antibodies, anti-CD20 antibodies, anti-CD22 antibodies, anti-CD25 antibodies, anti-CD27 antibodies, anti-CD30 antibodies, anti-CD33 antibodies, anti-CD37 antibodies, anti-CD40 antibodies, anti-CD41 antibodies, anti-CD44v6 antibodies, anti-CD51 Antibodies, anti-CD52 antibody, anti-CD56 antibody, anti-CD70 antibody, anti-CD74 antibody, anti-CD79 antibody, anti-CD79b antibody, anti-CEACAM5 antibody, anti-c-Met antibody, anti-DLL3 antibody, anti-DPEP3 antibody, anti-E GFR antibody, anti-EGFRvIII antibody, anti-ENPP3 antibody, anti-EpCAM antibody, anti-EphA4 antibody, anti-FGFR2 antibody, anti-FGFR3 antibody, anti-FTL3 antibody, anti-folate receptor α antibody, anti-glypican-3 antibody, anti- gpNMB antibody, anti-HER2 antibody, anti-HER3 antibody, anti-IL-3RA antibody, anti-LAMP1 antibody, anti-LIV-1 antibody, anti-LRRC15 antibody, anti-Ly6E antibody, anti-mesothelin antibody, anti-MUC-16 antibody, anti-N aPi2b antibody, anti-Nectin-4 antibody, anti-CD352 antibody, anti-P-cadherin antibody, anti-PMSA antibody, anti-protein tyrosine kinase 7 antibody, anti-SLITRK antibody, anti-STEAP1 antibody, anti-CD138 antibody , anti-tissue factor antibody, anti-CD71 antibody, anti-TIM-1 antibody, anti-Trop2 antibody, anti-5T4 antibody, anti-B7-H3 antibody, anti-CD163 macrophage receptor antibody, anti-CD38 antibody, anti-CD48 antibody, anti-cKit antibody, anti-guanylate cyclase C antibody, anti-gastrin releasing peptide antibody, anti-solute transporter antibody, anti-tumor-associated MUC-1 antibody, anti-GD2 antibody, anti-α4β7 integrin antibody, or anti-embodiin antibody. Another embodiment of the mAb includes anti-CD19 antibody, anti-CD20 antibody, anti-CD22 antibody, anti-CD30 antibody, anti-CD33 antibody, anti-CD52 antibody, anti-CD70 antibody, anti-CD79b antibody, anti-CEACAM5 antibody, anti-EGFR antibody, anti-EGFRvIII antibody, anti-gpNMB antibody, anti-HER2 antibody, anti-mesothelin antibody, anti-CD138 antibody, anti-CD38 antibody, or anti-GD2 antibody.Another embodiment of the mAb includes an anti-CD19 antibody, an anti-CD20 antibody, an anti-CD22 antibody, an anti-CD30 antibody, an anti-CD33 antibody, an anti-CD52 antibody, an anti-CD79b antibody, an anti-CEACAM5 antibody, an anti-EGFR antibody, an anti-EGFRvIII antibody, an anti-gpNMB antibody, an anti-HER2 antibody, an anti-mesothelin antibody, or an anti-CD138 antibody.

[0094] Here, the "antibody" may be any antibody that contains at least a heavy chain variable domain and a light chain variable domain, and may be a complete antibody or a fragment of a complete antibody that is an antigen-binding fragment having an antigen recognition site. A complete antibody has two full-length light chains and two full-length heavy chains, and each light chain and heavy chain are linked by a disulfide bond. Complete antibodies include IgA, IgD, IgE, IgM, and IgG, and IgG includes IgG1, IgG2, IgG3, and IgG4 as subtypes. In addition, the antibody is preferably a monoclonal antibody. The antibody moiety and the drug moiety are bound via sulfhydryl groups obtained by reducing disulfide bonds in the antibody.

[0095] "AMG595 antibody" refers to an anti-EGFRvIII antibody obtained by the method described in Mol. Cancer Ther., 2015, 14, 1614-1624.

[0096] In one embodiment, Z may be a group represented by formula (Z-1), formula (Z-2) or formula (Z-3), and Q may be an unsubstituted phenyl group or a group represented by formula (Q-1).

[0097] In one embodiment, Z' may be a group represented by formula (Z-4) or formula (Z-5), and Q may be an unsubstituted phenyl group or a group represented by formula (Q-1).

[0098] One embodiment of the antibody-drug conjugate of the present invention includes the following (2-1-A). (2-1-A) In formula (2-1), the mAb is brentuximab, q is an integer from 1 to 8; b is 2, 3, 4 or 5; Z is a group represented by formula (Z-1), formula (Z-2) or formula (Z-3), n is an integer of 0 or 1; Q is an unsubstituted phenyl group or a group represented by formula (Q-1), R 1a is a methyl group, R 1b is a hydrogen atom, R 2 But -(CH2) u -COR 4 and u is an integer of 1 or 2; AA is Glu or Asp; R 3 is -OH or -(AB) p and R 4 is -OH or -(AB) p and p is an integer of 1 or 2; AB represents Glu or Asp, and when there are a plurality of AB, each AB may be the same or different, and AB are bonded to each other via an amide bond; R 3 or R 4 Ga-(AB) p when the sum of n and p is 1 or 2, An antibody-drug conjugate or a pharma- ceutically acceptable salt thereof.

[0099] One embodiment of the antibody-drug conjugate of the present invention includes the following (2-1-B). (2-1-B) In formula (2-1), the mAb is trastuzumab; q is an integer from 1 to 8; b is 2, 3, 4 or 5; Z is a group represented by formula (Z-1), formula (Z-2) or formula (Z-3), n is an integer of 0 or 1; Q is an unsubstituted phenyl group or a group represented by formula (Q-1), R 1a is a methyl group, R 1b is a hydrogen atom, R 2 But -(CH2) u -COR 4 and u is an integer of 1 or 2; AA is Glu or Asp; R 3 is -OH or -(AB) p and R 4 is -OH or -(AB) p and p is an integer of 1 or 2; AB represents Glu or Asp, and when there are a plurality of AB, each AB may be the same or different, and AB are bonded to each other via an amide bond; R 3 or R 4 Ga-(AB) p when the sum of n and p is 1 or 2, An antibody-drug conjugate or a pharma- ceutically acceptable salt thereof.

[0100] One embodiment of the antibody-drug conjugate of the present invention includes the following (2-1-C). (2-1-C) In formula (2-1), the mAb is gemtuzumab; q is an integer from 1 to 8; b is 2, 3, 4 or 5; Z is a group represented by formula (Z-1), formula (Z-2) or formula (Z-3), n is an integer of 0 or 1; Q is an unsubstituted phenyl group or a group represented by formula (Q-1), R 1a is a methyl group, R 1b is a hydrogen atom, R 2But -(CH2) u -COR 4 and u is an integer of 1 or 2; AA is Glu or Asp; R 3 is -OH or -(AB) p and R 4 is -OH or -(AB) p and p is an integer of 1 or 2; AB represents Glu or Asp, and when there are a plurality of AB, each AB may be the same or different, and AB are bonded to each other via an amide bond; R 3 or R 4 Ga-(AB) p when the sum of n and p is 1 or 2, An antibody-drug conjugate or a pharma- ceutically acceptable salt thereof.

[0101] One embodiment of the antibody-drug conjugate of the present invention includes the following (2-1-D). (2-1-D) In formula (2-1), the mAb is inotuzumab; q is an integer from 1 to 8; b is 2, 3, 4 or 5; Z is a group represented by formula (Z-1), formula (Z-2) or formula (Z-3), n is an integer of 0 or 1; Q is an unsubstituted phenyl group or a group represented by formula (Q-1), R 1a is a methyl group, R 1b is a hydrogen atom, R 2 But -(CH2) u -COR 4 and u is an integer of 1 or 2; AA is Glu or Asp; R 3is -OH or -(AB) p and R 4 is -OH or -(AB) p and p is an integer of 1 or 2; AB represents Glu or Asp, and when there are a plurality of AB, each AB may be the same or different, and AB are bonded to each other via an amide bond; R 3 or R 4 Ga-(AB) p when the sum of n and p is 1 or 2, An antibody-drug conjugate or a pharma- ceutically acceptable salt thereof.

[0102] One embodiment of the antibody-drug conjugate of the present invention includes the following (2-1-E). (2-1-E) In formula (2-1), the mAb is rituximab; q is an integer from 1 to 8; b is 2, 3, 4 or 5; Z is a group represented by formula (Z-1), formula (Z-2) or formula (Z-3), n is an integer of 0 or 1; Q is an unsubstituted phenyl group or a group represented by formula (Q-1), R 1a is a methyl group, R 1b is a hydrogen atom, R 2 But -(CH2) u -COR 4 and u is an integer of 1 or 2; AA is Glu or Asp; R 3 is -OH or -(AB) p and R 4 is -OH or -(AB) p and p is an integer of 1 or 2; AB represents Glu or Asp, and when there are a plurality of AB, each AB may be the same or different, and AB are bonded to each other via an amide bond; R 3 or R 4 Ga-(AB) p when the sum of n and p is 1 or 2, An antibody-drug conjugate or a pharma- ceutically acceptable salt thereof.

[0103] One embodiment of the antibody-drug conjugate of the present invention includes the following (2-1-F). (2-1-F) In formula (2-1), the mAb is cetuximab; q is an integer from 1 to 8; b is 2, 3, 4 or 5; Z is a group represented by formula (Z-1), formula (Z-2) or formula (Z-3), n is an integer of 0 or 1; Q is an unsubstituted phenyl group or a group represented by formula (Q-1), R 1a is a methyl group, R 1b is a hydrogen atom, R 2 But -(CH2) u -COR 4 and u is an integer of 1 or 2; AA is Glu or Asp; R 3 is -OH or -(AB) p and R 4 is -OH or -(AB) p and p is an integer of 1 or 2; AB represents Glu or Asp, and when there are a plurality of AB, each AB may be the same or different, and AB are bonded to each other via an amide bond; R 3 or R 4 Ga-(AB)p when the sum of n and p is 1 or 2, An antibody-drug conjugate or a pharma- ceutically acceptable salt thereof.

[0104] One embodiment of the antibody-drug conjugate of the present invention includes the following (2-1-G). (2-1-G) In formula (2-1), the mAb is cortuximab; q is an integer from 1 to 8; b is 2, 3, 4 or 5; Z is a group represented by formula (Z-1), formula (Z-2) or formula (Z-3), n is an integer of 0 or 1, Q is an unsubstituted phenyl group or a group represented by formula (Q-1), R 1a is a methyl group, R 1b is a hydrogen atom, R 2 But -(CH2) u -COR 4 and u is an integer of 1 or 2; AA is Glu or Asp; R 3 is -OH or -(AB) p and R 4 is -OH or -(AB) p and p is an integer of 1 or 2; AB represents Glu or Asp, and when there are a plurality of AB, each AB may be the same or different, and AB are bonded to each other via an amide bond; R 3 or R 4 Ga-(AB) p when the sum of n and p is 1 or 2, An antibody-drug conjugate or a pharma- ceutically acceptable salt thereof.

[0105] One embodiment of the antibody-drug conjugate of the present invention is shown below in (2-1-H). (2-1-H) In formula (2-1), the mAb is denintuzumab; q is an integer from 1 to 8; b is 2, 3, 4 or 5; Z is a group represented by formula (Z-1), formula (Z-2) or formula (Z-3), n is an integer of 0 or 1; Q is an unsubstituted phenyl group or a group represented by formula (Q-1), R 1a is a methyl group, R 1b is a hydrogen atom, R 2 But -(CH2) u -COR 4 and u is an integer of 1 or 2; AA is Glu or Asp; R 3 is -OH or -(AB) p and R 4 is -OH or -(AB) p and p is an integer of 1 or 2; AB represents Glu or Asp, and when there are a plurality of AB, each AB may be the same or different, and AB are bonded to each other via an amide bond; R 3 or R 4 Ga-(AB) p when the sum of n and p is 1 or 2, An antibody-drug conjugate or a pharma- ceutically acceptable salt thereof.

[0106] One embodiment of the antibody-drug conjugate of the present invention includes the following (2-1-I). (2-1-I) In formula (2-1), the mAb is alemtuzumab; q is an integer from 1 to 8; b is 2, 3, 4 or 5; Z is a group represented by formula (Z-1), formula (Z-2) or formula (Z-3), n is an integer of 0 or 1; Q is an unsubstituted phenyl group or a group represented by formula (Q-1), R 1a is a methyl group, R 1b is a hydrogen atom, R 2 But -(CH2) u -COR 4 and u is an integer of 1 or 2; AA is Glu or Asp; R 3 is -OH or -(AB) p and R 4 is -OH or -(AB) p and p is an integer of 1 or 2; AB represents Glu or Asp, and when there are a plurality of AB, each AB may be the same or different, and AB are bonded to each other via an amide bond; R 3 or R 4 Ga-(AB) p when the sum of n and p is 1 or 2, An antibody-drug conjugate or a pharma- ceutically acceptable salt thereof.

[0107] One embodiment of the antibody-drug conjugate of the present invention includes the following (2-1-J). (2-1-J) In formula (2-1), the mAb is anetumab; q is an integer from 1 to 8; b is 2, 3, 4 or 5; Z is a group represented by formula (Z-1), formula (Z-2) or formula (Z-3), n is an integer of 0 or 1; Q is an unsubstituted phenyl group or a group represented by formula (Q-1), R 1a is a methyl group, R 1b is a hydrogen atom, R 2 But -(CH2) u -COR 4 and u is an integer of 1 or 2; AA is Glu or Asp; R 3 is -OH or -(AB) p and R 4 is -OH or -(AB) p and p is an integer of 1 or 2; AB represents Glu or Asp, and when there are a plurality of AB, each AB may be the same or different, and AB are bonded to each other via an amide bond; R 3 or R 4 Ga-(AB) p when the sum of n and p is 1 or 2, An antibody-drug conjugate or a pharma- ceutically acceptable salt thereof.

[0108] One embodiment of the antibody-drug conjugate of the present invention includes the following (2-1-K). (2-1-K) In formula (2-1), the mAb is polatuzumab; q is an integer from 1 to 8; b is 2, 3, 4 or 5; Z is a group represented by formula (Z-1), formula (Z-2) or formula (Z-3), n is an integer of 0 or 1; Q is an unsubstituted phenyl group or a group represented by formula (Q-1), R 1a is a methyl group, R 1b is a hydrogen atom, R2 But -(CH2) u -COR 4 and u is an integer of 1 or 2; AA is Glu or Asp; R 3 is -OH or -(AB) p and R 4 is -OH or -(AB) p and p is an integer of 1 or 2; AB represents Glu or Asp, and when there are a plurality of AB, each AB may be the same or different, and AB are bonded to each other via an amide bond; R 3 or R 4 Ga-(AB) p when the sum of n and p is 1 or 2, An antibody-drug conjugate or a pharma- ceutically acceptable salt thereof.

[0109] One embodiment of the antibody-drug conjugate of the present invention includes the following (2-1-L). (2-1-L) In formula (2-1), the mAb is vadastuximab; q is an integer from 1 to 8; b is 2, 3, 4 or 5; Z is a group represented by formula (Z-1), formula (Z-2) or formula (Z-3), n is an integer of 0 or 1; Q is an unsubstituted phenyl group or a group represented by formula (Q-1), R 1a is a methyl group, R 1b is a hydrogen atom, R 2 But -(CH2) u -COR 4 and u is an integer of 1 or 2; AA is Glu or Asp; R3 is -OH or -(AB) p and R 4 is -OH or -(AB) p and p is an integer of 1 or 2; AB represents Glu or Asp, and when there are a plurality of AB, each AB may be the same or different, and AB are bonded to each other via an amide bond; R 3 or R 4 Ga-(AB) p when the sum of n and p is 1 or 2, An antibody-drug conjugate or a pharma- ceutically acceptable salt thereof.

[0110] One embodiment of the antibody-drug conjugate of the present invention includes the following (2-1-M). (2-1-M) In formula (2-1), the mAb is glembatumumab; q is an integer from 1 to 8; b is 2, 3, 4 or 5; Z is a group represented by formula (Z-1), formula (Z-2) or formula (Z-3), n is an integer of 0 or 1; Q is an unsubstituted phenyl group or a group represented by formula (Q-1), R 1a is a methyl group, R 1b is a hydrogen atom, R 2 But -(CH2) u -COR 4 and u is an integer of 1 or 2; AA is Glu or Asp; R 3 is -OH or -(AB) p and R 4 is -OH or -(AB) p and p is an integer of 1 or 2; AB represents Glu or Asp, and when there are a plurality of AB, each AB may be the same or different, and AB are bonded to each other via an amide bond; R 3 or R 4 Ga-(AB) p when the sum of n and p is 1 or 2, An antibody-drug conjugate or a pharma- ceutically acceptable salt thereof.

[0111] One embodiment of the antibody-drug conjugate of the present invention includes the following (2-1-N). (2-1-N) In formula (2-1), the mAb is indatuximab; q is an integer from 1 to 8; b is 2, 3, 4 or 5; Z is a group represented by formula (Z-1), formula (Z-2) or formula (Z-3), n is an integer of 0 or 1; Q is an unsubstituted phenyl group or a group represented by formula (Q-1), R 1a is a methyl group, R 1b is a hydrogen atom, R 2 But -(CH2) u -COR 4 and u is an integer of 1 or 2; AA is Glu or Asp; R 3 is -OH or -(AB) p and R 4 is -OH or -(AB) p and p is an integer of 1 or 2; AB represents Glu or Asp, and when there are a plurality of AB, each AB may be the same or different, and AB are bonded to each other via an amide bond; R 3 or R4 Ga-(AB) p when the sum of n and p is 1 or 2, An antibody-drug conjugate or a pharma- ceutically acceptable salt thereof.

[0112] One embodiment of the antibody-drug conjugate of the present invention includes the following (2-1-O). (2-1-O) In formula (2-1), the mAb is depatuxizumab; q is an integer from 1 to 8; b is 2, 3, 4 or 5; Z is a group represented by formula (Z-1), formula (Z-2) or formula (Z-3), n is an integer of 0 or 1; Q is an unsubstituted phenyl group or a group represented by formula (Q-1), R 1a is a methyl group, R 1b is a hydrogen atom, R 2 But -(CH2) u -COR 4 and u is an integer of 1 or 2; AA is Glu or Asp; R 3 is -OH or -(AB) p and R 4 is -OH or -(AB) p and p is an integer of 1 or 2; AB represents Glu or Asp, and when there are a plurality of AB, each AB may be the same or different, and AB are bonded to each other via an amide bond; R 3 or R 4 Ga-(AB) p when the sum of n and p is 1 or 2, An antibody-drug conjugate or a pharma- ceutically acceptable salt thereof.

[0113] One embodiment of the antibody-drug conjugate of the present invention includes the following (2-1-P). (2-1-P) In formula (2-1), the mAb is laprituximab; q is an integer from 1 to 8; b is 2, 3, 4 or 5; Z is a group represented by formula (Z-1), formula (Z-2) or formula (Z-3), n is an integer of 0 or 1; Q is an unsubstituted phenyl group or a group represented by formula (Q-1), R 1a is a methyl group, R 1b is a hydrogen atom, R 2 But -(CH2) u -COR 4 and u is an integer of 1 or 2; AA is Glu or Asp; R 3 is -OH or -(AB) p and R 4 is -OH or -(AB) p and p is an integer of 1 or 2; AB represents Glu or Asp, and when there are a plurality of AB, each AB may be the same or different, and AB are bonded to each other via an amide bond; R 3 or R 4 Ga-(AB) p when the sum of n and p is 1 or 2, An antibody-drug conjugate or a pharma- ceutically acceptable salt thereof.

[0114] One embodiment of the antibody-drug conjugate of the present invention includes the following (2-1-P). (2-1-Q) In formula (2-1), the mAb is labetuzumab; q is an integer from 1 to 8; b is 2, 3, 4 or 5; Z is a group represented by formula (Z-1), formula (Z-2) or formula (Z-3), n is an integer of 0 or 1; Q is an unsubstituted phenyl group or a group represented by formula (Q-1), R 1a is a methyl group, R 1b is a hydrogen atom, R 2 But -(CH2) u -COR 4 and u is an integer of 1 or 2; AA is Glu or Asp; R 3 is -OH or -(AB) p and R 4 is -OH or -(AB) p and p is an integer of 1 or 2; AB represents Glu or Asp, and when there are a plurality of AB, each AB may be the same or different, and AB are bonded to each other via an amide bond; R 3 or R 4 Ga-(AB) p when the sum of n and p is 1 or 2, An antibody-drug conjugate or a pharma- ceutically acceptable salt thereof.

[0115] One embodiment of the antibody-drug conjugate of the present invention includes the following (2-2-A). (2-2-A) In formula (2-2), the mAb is brentuximab, q is an integer from 1 to 8; h is 2, 3, 4 or 5; Z' is a group represented by formula (Z-4) or formula (Z-5), Q is an unsubstituted phenyl group or a group represented by formula (Q-1), R 1a is a methyl group, R 1b is a hydrogen atom, AA is Glu or Asp; n is 0, 1, or 2; An antibody-drug conjugate or a pharma- ceutically acceptable salt thereof.

[0116] In general, antibody-drug conjugates can be produced and analyzed by any technique known to those skilled in the art, such as those described in Antibody-Drug Conjugates (edited by Laurent Ducry, published by Humana Press, 2013).

[0117] The antibody-drug conjugate of the present invention can be formed, for example, by reducing disulfide bonds in the antibody to sulfhydryl groups and reacting the sulfhydryl groups with a hemiasterlin derivative.

[0118] In the antibody-drug conjugate of the present invention, the antibody is metabolized in a target cell (antigen-expressing cell) to release the drug moiety or a structure including a part of the antibody (antibody fragment) and the drug moiety. For example, Doronina SO et al., 2006, Bioconjugate Chem. 17:114-124, describes that the Cys-drug moiety of the antibody-drug conjugate is released within the cell by the metabolism of the antibody.

[0119] Since the antibody-drug conjugate of the present invention has a structure that is difficult to be cleaved by blood peptidases, the bound state between the drug moiety and the antibody moiety can be maintained even in the blood, and the release of free drug into the blood is suppressed. Therefore, the antibody-drug conjugate of the present invention reduces the release of the drug into the blood before it reaches the target cells.

[0120] In addition, since the drug moiety of the antibody-drug conjugate of the present invention has low cell membrane permeability, once released inside a target cell, the drug moiety is unlikely to migrate outside the cell, and therefore the drug exerts its efficacy only inside the target cell, and the drug can exert cytotoxicity specifically on antigen-expressing cells.

[0121] Furthermore, even if dissociation of the thiosuccinimide site in the body occurs in the antibody-drug conjugate of the present invention as reported in Non-Patent Documents 7 and 8, and the drug moiety is released in the blood before the antibody-drug conjugate reaches the target cell, the drug moiety of the antibody-drug conjugate of the present invention has low cell membrane permeability, so that the drug is unlikely to passively diffuse into normal cells and is rapidly metabolized and excreted.

[0122] As described above, the antibody-drug conjugate of the present invention can impart cytotoxicity by the drug (hemiacetalin derivative) specifically to antigen-expressing cells, and is expected to cause fewer side effects due to systemic exposure.

[0123] The "salt" is a suitable salt of the hemiasterlin derivative of the present invention and a salt acceptable as a pharmaceutical raw material, preferably a conventional non-toxic salt. As the "salt", for example, an acid addition salt such as an organic acid salt (e.g., acetate, trifluoroacetate, maleate, fumarate, citrate, tartrate, methanesulfonate, benzenesulfonate, formate, or p-toluenesulfonate, etc.) and an inorganic acid salt (e.g., hydrochloride, hydrobromide, hydroiodide, sulfate, nitrate, or phosphate, etc.), a salt with an amino acid (e.g., arginine, aspartic acid, or glutamic acid, etc.), a metal salt such as an alkali metal salt (e.g., sodium salt, potassium salt, etc.) and an alkaline earth metal salt (e.g., calcium salt, magnesium salt, etc.), an ammonium salt, or an organic base salt (e.g., trimethylamine salt, triethylamine salt, pyridine salt, picoline salt, dicyclohexylamine salt, or N,N'-dibenzylethylenediamine salt, etc.), etc., and other suitable salts can be appropriately selected by those skilled in the art.

[0124] The "pharmaceutical acceptable salt" includes acid addition salts and base addition salts. For example, the acid addition salts include inorganic acid salts such as hydrochloride, hydrobromide, sulfate, hydroiodide, nitrate, and phosphate, and organic acid salts such as citrate, oxalate, phthalate, fumarate, maleate, succinate, malate, acetate, formate, propionate, benzoate, trifluoroacetate, methanesulfonate, benzenesulfonate, p-toluenesulfonate, and camphorsulfonate. In addition, the base addition salts include inorganic base salts such as sodium salt, potassium salt, calcium salt, magnesium salt, barium salt, and aluminum salt, and organic base salts such as trimethylamine, triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, tromethamine [tris(hydroxymethyl)methylamine], tert-butylamine, cyclohexylamine, dicyclohexylamine, and N,N-dibenzylethylamine. Furthermore, the "pharmaceutically acceptable salt" also includes salts with basic or acidic amino acids such as arginine, lysine, ornithine, aspartic acid, glutamic acid, etc. (amino acid salts).

[0125] When it is desired to obtain a salt of the hemiasterlin derivative or antibody-drug conjugate of the present invention, if the target compound is obtained in the form of a salt, it can be purified as is. If the target compound is obtained in the free form, it can be dissolved or suspended in an appropriate organic solvent and an acid or base can be added to form a salt by a conventional method.

[0126] The hemiasterlin derivative and antibody-drug conjugate of the present invention may exist in the form of a hydrate and / or a solvate with various solvents (such as an ethanol solvate), and these hydrates and / or solvates are also included in the hemiasterlin derivative and antibody-drug conjugate of the present invention. Furthermore, the present invention also includes all forms of crystal forms of the hemiasterlin derivative and antibody-drug conjugate of the present invention.

[0127] Among the hemiasterlin derivatives and antibody-drug conjugates of the present invention, there may exist optical isomers based on optically active centers, atropisomers based on axial or planar chirality arising from restricted intramolecular rotation, and all other stereoisomers, tautomers, and geometric isomers, and all possible isomers, including these, are included in the scope of the present invention.

[0128] In particular, optical isomers and atropisomers can be obtained as racemates, or as optically active isomers when optically active starting materials or intermediates are used. If necessary, at an appropriate stage of the following production method, the corresponding racemates of the starting materials, intermediates or final products can be physically or chemically optically resolved into their optical antipodes by known separation methods such as a method using an optically active column or a fractional crystallization method. Specifically, for example, in the diastereomeric method, two diastereomers are formed from a racemate by a reaction using an optically active resolving agent. These different diastereomers generally have different physical properties, and therefore can be optically resolved by known methods such as fractional crystallization.

[0129] The method for producing the hemiasterlin derivative of the present invention is described below. The hemiasterlin derivative of the present invention represented by formula (1-1) or (1-2) can be produced, for example, by the following Production Methods A to I, L to P, T, U, or W.

[0130] Manufacturing method A Z is a group represented by formula (Z-1), Q is a group represented by formula (Q-1), and R 1a is a methyl group, and R 1b is a hydrogen atom or a methyl group, and R 2 Ga-(CH2) u In the case of -COOH, the compound represented by formula (1-1) can be produced, for example, by the following production method. [ka] (Wherein, AA, u, b and n are as defined in Section 2, R a , Rb , R x , R y and R Z are each independently 1-6 represents an alkyl group or a benzyl group, P X means a protecting group for an amino group.)

[0131] Above P X Examples of the protecting group for the amino group represented by the formula (I) include protecting groups described in Protective Groups in Organic Synthesis (Theodora W. Greene, Peter G. M. Wuts, published by John Wiley & Sons, Inc., 1999).

[0132] Compound a1 can be prepared by the method described in, for example, J. Med. Chem., 2007, 50, 4329-4339, etc., or can be purchased as a commercial product. Compound a15 can be prepared by the method described in, for example, Tetrahedron Lett., 1997, 38, 317-320, etc., or can be purchased as a commercial product.

[0133] [A-1 process] Compound a2 can be produced by reacting compound a1 with various methylating reagents in a suitable solvent in the presence of a suitable base. Examples of the methylating reagent include methyl halides, and preferably methyl iodide, methyl bromide, and methyl chloride. Examples of the base include potassium hexamethyldisilazide. Examples of the solvent include tetrahydrofuran. The reaction time is usually 5 minutes to 48 hours, and preferably 10 minutes to 2 hours. The reaction temperature is usually -78°C to 100°C, and preferably -78°C to 10°C. This step can be carried out in accordance with the method described in J. Nat. Prod. 2003, 66, 183-199, etc.

[0134] [A-2 process] Compound a3 can be produced from compound a2 according to the method described in step A-1 above.

[0135] [A-3 process] Compound a4 can be produced by reacting compound a3 with a suitable reducing agent in a suitable solvent. The reducing agent is appropriately selected from reducing agents used in ordinary organic synthesis reactions, and preferably includes diisobutylaluminum hydride. The solvent is preferably diethyl ether. The reaction time is usually 5 minutes to 48 hours, and preferably 10 minutes to 24 hours. The reaction temperature is usually -78°C to 100°C, and preferably -78°C to 50°C. This step can be carried out in accordance with the method described in J. Nat. Prod. 2003, 66, 183-199, etc.

[0136] [A-4 process] Compound a5 can be produced by oxidizing compound a4 using a suitable oxidizing agent in a suitable solvent. The oxidizing agent can be appropriately selected from oxidizing agents used in normal organic synthesis reactions, and preferably includes tetrapropylammonium perruthenate. The solvent preferably includes dichloromethane. The reaction time is usually 5 minutes to 72 hours, preferably 30 minutes to 24 hours. The reaction temperature is usually -78°C to 100°C, preferably -78°C to 50°C. This step can be carried out in accordance with the method described in J. Nat. Prod. 2003, 66, 183-199, etc.

[0137] [A-5 process] Compound a6 can be produced by α-aminocyanating the aldehyde of compound a5 in a suitable solvent. Preferred examples of the solvent include toluene and dichloromethane. The reaction time is usually 5 minutes to 96 hours, preferably 24 hours to 72 hours. The reaction temperature is usually 0°C to 200°C, preferably 0°C to 100°C. This step can be carried out in accordance with the method described in Org. Lett. 2002, 4, 695-697, etc.

[0138] [A-6 process] Compound a7 can be produced by using a suitable oxidizing agent in a suitable solvent in the presence or absence of a suitable base from compound a6. The oxidizing agent can be appropriately selected from oxidizing agents used in normal organic synthesis reactions, and preferably includes hydrogen peroxide. The base preferably includes potassium carbonate. The solvent preferably includes methanol. The reaction time is usually 5 minutes to 72 hours, preferably 30 minutes to 24 hours. The reaction temperature is usually 0°C to 200°C, preferably 0°C to 60°C. This step can be carried out in accordance with the method described in J. Org. Chem. 2001, 66, 7355-7364, etc.

[0139] [A-7 process] Compound a8 can be produced by reducing compound a7 in a suitable solvent in the presence of a suitable catalyst using a reducing agent. The reducing agent can be appropriately selected from reducing agents used in ordinary organic synthesis reactions, and preferably includes hydrogen, a salt of formic acid such as ammonium formate, or hydrazine. The catalyst includes transition metals such as palladium, nickel, rhodium, cobalt, platinum, salts thereof, complexes thereof, or the above transition metals supported on a carrier such as a polymer. The solvent includes preferably ethanol or methanol. The reaction time is usually 5 minutes to 72 hours, preferably 30 minutes to 24 hours. The reaction temperature is usually 0°C to 200°C, preferably 0°C to 100°C. This step can be carried out in accordance with the method described in J. Org. Chem. 2001, 66, 7355-7364, etc.

[0140] [A-8 process] Compound a9 is prepared by converting the amino group of compound a8 into a protecting group P X This step can be carried out in accordance with the method described in Protective Groups in Organic Synthesis (Theodora W. Greene, Peter GM Wuts, John Wiley & Sons, Inc., 1999).

[0141] [A-9 process] Compound A11 can be produced by reacting Compound A9 with various acylating reagents (e.g., Compound A10) in a suitable solvent in the presence or absence of a suitable base. Examples of the acylating reagent include carboxylic acid halides and carboxylic acid anhydrides, and preferably di-tert-butyl dicarbonate. A preferred example of the base is diisopropylethylamine. A preferred example of the solvent is chloroform. The reaction time is usually 5 minutes to 72 hours, and preferably 30 minutes to 24 hours. The reaction temperature is usually 0°C to 200°C, and preferably 0°C to 50°C.

[0142] [A-10 process] Compound a12 can be produced by reacting compound a11 with an appropriate alkali metal alkoxide in an appropriate solvent. The alkali metal alkoxide can be appropriately selected from alkali metal alkoxides used in normal organic synthesis reactions, and preferably includes lithium methoxide or lithium ethoxide. The solvent is preferably methanol or ethanol. The reaction time is usually 5 minutes to 72 hours, preferably 30 minutes to 6 hours. The reaction temperature is usually -78°C to 200°C, preferably -78°C to 50°C.

[0143] [A-11 process] Compound a13 can be produced by reacting compound a12 with various methylating reagents in the presence of a suitable base in a suitable solvent. Examples of the methylating reagent include methyl halides, and preferably methyl iodide, methyl bromide, and methyl chloride. Examples of the base include sodium hydride. Examples of the solvent include tetrahydrofuran. The reaction time is usually 5 minutes to 48 hours, and preferably 10 minutes to 2 hours. The reaction temperature is usually -78°C to 100°C, and preferably -78°C to 10°C.

[0144] [A-12 process] Compound a14 can be produced by hydrolyzing the ester of compound a13 in the presence of a suitable base in a suitable solvent. The base is preferably lithium hydroxide. The solvent is preferably water or methanol. The reaction time is usually 5 minutes to 72 hours, preferably 30 minutes to 24 hours. The reaction temperature is usually 0°C to 200°C, preferably 0°C to 100°C.

[0145] [A-13 process] Compound a16 can be produced by condensing compound a14 and compound a15 in a suitable solvent in the presence of a suitable base using various condensing agents. As the condensing agent, various condensing agents used in normal organic synthesis reactions can be used, and preferably (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide or bromotripyrrolidinophosphonium hexafluorophosphate can be used. If necessary, a carbonyl activating agent such as 1-hydroxybenzotriazole can be used together to improve the efficiency of the condensation reaction. As the base, preferably diisopropylethylamine can be used. As the solvent, preferably N,N-dimethylformamide can be used. The reaction time is usually 5 minutes to 72 hours, preferably 30 minutes to 24 hours. The reaction temperature is usually -78°C to 200°C, preferably 0°C to 100°C. This step can be carried out in accordance with the method described in Tetrahedron Lett., 1997, 38, 317-320, etc.

[0146] [A-14 process] Compound a17 can be produced by hydrolyzing the ester of compound a16 in accordance with the method described in step A-12 above. This step can be carried out in accordance with the method described in Tetrahedron Lett., 1997, 38, 317-320, etc.

[0147] [A-15 process] Compound a18 can be produced by reacting compound a17 with N-hydroxysuccinimide in a suitable solvent in the presence of a suitable base using various condensing agents. As the condensing agent, various condensing agents used in normal organic synthesis reactions can be used, and preferably (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide or bromotripyrrolidinophosphonium hexafluorophosphate can be used. If necessary, a carbonyl activating agent such as 1-hydroxybenzotriazole can be used together to improve the efficiency of the reaction. As the base, preferably, diisopropylethylamine can be used. As the solvent, preferably, N,N-dimethylformamide can be used. The reaction time is usually 5 minutes to 72 hours, preferably 30 minutes to 24 hours. The reaction temperature is usually -78°C to 200°C, preferably 0°C to 100°C.

[0148] [A-16 process] Compound a19 can be produced by reacting compound a18 with an ester of an amino acid or peptide in the presence of a suitable base in a suitable solvent. A preferred base is diisopropylethylamine. A preferred solvent is N,N-dimethylformamide. The reaction time is usually 5 minutes to 72 hours, preferably 30 minutes to 24 hours. The reaction temperature is usually 0°C to 200°C, preferably 0°C to 100°C.

[0149] [A-17 process] Compound a20 can be produced by condensing compound a19 with an aminoalkylmaleimide compound according to the method described in step A-13 above.

[0150] [A-18 process] Compound A1 is a compound a20 having a protecting group P XThis step can be carried out in accordance with the method described in Protective Groups in Organic Synthesis (Theodora W. Greene, Peter GM Wuts, John Wiley & Sons, Inc., 1999), or the like. Also, (AA) n When has an ester or a protected amino group, hydrolysis of the ester and deprotection of the protecting group of the amino group can be carried out in this deprotection step, if necessary.

[0151] [A-19 process] Compound A2 can be produced by reacting Compound A1 with formaldehyde in a suitable solvent together with a suitable reducing agent. As the solvent, acetonitrile is preferable. As the reducing agent, various reducing agents used in normal organic synthesis reactions can be used, and sodium triacetoxyborohydride is preferable. The reaction time is usually 5 minutes to 72 hours, preferably 30 minutes to 24 hours. The reaction temperature is usually 0°C to about 200°C, preferably 0°C to 100°C.

[0152] Manufacturing method B Z is a group represented by formula (Z-1), Q is an unsubstituted phenyl group, and R 1a is a methyl group, and R 1b is a hydrogen atom or a methyl group, and R 2 Ga-(CH2) u In the case of -COOH, the compound represented by formula (1-1) can be produced, for example, by the following production method. [ka] (Wherein, AA, u, b and n are as defined in Section 2, R a , R b and R x is C 1-6 represents an alkyl group or a benzyl group, P Xmeans a protecting group for an amino group.)

[0153] Compound b1 can be purchased, for example, as a commercially available product. Compound b11 can be prepared, for example, by the method described in Tetrahedron Lett., 1997, 38, 317-320, or can be purchased as a commercially available product.

[0154] [B-1 process] Compound b2 can be produced by reacting compound b1 with benzene in the presence of various Lewis acids. Examples of Lewis acids include boron halides, aluminum halides, gallium halides, iron halides, titanium halides, and the like, and preferably aluminum chloride, iron chloride, and the like. The reaction time is usually 5 minutes to 48 hours, and preferably 30 minutes to 4 hours. The reaction temperature is usually -78°C to 200°C, and preferably 50°C to 150°C. This step can be carried out in accordance with the method described in J. Nat. Prod. 2003, 66, 183-199, etc.

[0155] [B-2 process] Compound b3 can be produced by reacting compound b2 with various carboxylic acid halides in a suitable solvent in the presence of a suitable base, and then reacting with an alkali metal 4-alkyl-2-oxazolidinone. As the base, triethylamine or diisopropylethylamine is preferably used. As the solvent, tetrahydrofuran is preferably used. As the carboxylic acid halides, for example, carboxylic acid chlorides and the like are included, and preferably pivaloyl chloride and the like are included. As the alkali metal 4-alkyl-2-oxazolidinones, 4-alkyl-2-oxazolidinone lithium, 4-alkyl-2-oxazolidinone sodium and the like are included, and preferably 4-isopropyl-2-oxazolidinone lithium is included. The reaction time is usually 5 minutes to 48 hours, and preferably 10 minutes to 24 hours. The reaction temperature is usually -78°C to 100°C, and preferably -78°C to 50°C. This step can be carried out in accordance with the method described in J. Nat. Prod. 2003, 66, 183-199, etc.

[0156] [B-3 process] Compound b4 can be produced by reacting compound b3 with various azidation reagents in a suitable solvent in the presence of a suitable base. Examples of the azidation reagent include sodium azide, trimethylsilyl azide, diphenylphosphoryl azide, and the like, and preferably trimethylsilyl azide. Examples of the base include potassium hexamethyldisilazide. Examples of the solvent include tetrahydrofuran. The reaction time is usually 5 minutes to 72 hours, and preferably 30 minutes to 24 hours. The reaction temperature is usually -78°C to 200°C, and preferably -78°C to 75°C. This step can be carried out in accordance with the method described in J. Nat. Prod. 2003, 66, 183-199, etc.

[0157] [B-4 process] Compound b5 can be produced from compound b4 according to the method described in step A-7 above.

[0158] [B-5 process] Compound b6 can be produced from compound b5 according to the method described in step A-8 above.

[0159] [B-6 process] Compound b7 can be produced by using a suitable oxidizing agent from compound b6 in a suitable solvent in the presence of a suitable base. The base is preferably lithium hydroxide. The solvent is preferably methanol, tetrahydrofuran or water. The oxidizing agent can be appropriately selected from oxidizing agents used in normal organic synthesis reactions, and preferably hydrogen peroxide. The reaction time is usually 5 minutes to 72 hours, preferably 30 minutes to 24 hours. The reaction temperature is usually 0°C to 200°C, preferably 0°C to 60°C. This step can be carried out in accordance with the method described in J. Nat. Prod. 2003, 66, 183-199, etc.

[0160] [B-7 process] Compound b8 can be produced by reacting compound b7 with various alkylating reagents in the presence of a suitable base in a suitable solvent. Examples of alkylating reagents include alkyl halides, and preferably alkyl iodides, alkyl bromides, and alkyl chlorides. Examples of bases include sodium carbonate and potassium carbonate. Examples of solvents include N,N-dimethylformamide. The reaction time is usually 5 minutes to 48 hours, and preferably 10 minutes to 2 hours. The reaction temperature is usually -78°C to 100°C, and preferably -10°C to 25°C. This step can be carried out according to the method described in Protective Groups in Organic Synthesis (Theodora W. Greene, Peter GM Wuts, published by John Wiley & Sons, Inc., 1999), and the like.

[0161] [B-8 process] Compound b9 can be produced from compound b8 according to the method described in step A-11 above.

[0162] [B-9 process] Compound b10 can be produced from compound b9 according to the method described in step A-12 above.

[0163] [B-10 process] Compound b12 can be produced from compound b10 and compound b11 according to the method described in step A-13 above.

[0164] [B-11 process] Compound b13 can be produced by hydrolyzing the ester of compound b12 in accordance with the method described in step A-12 above.

[0165] [B-12 process] Compound b14 can be produced from compound b13 according to the method described in step A-15 above.

[0166] [B-13 process] Compound b15 can be produced from compound b14 according to the method described in step A-16 above.

[0167] [B-14 process] Compound b16 can be produced from compound b15 according to the method described in step A-17 above.

[0168] [B-15 process] Compound B1 can be produced from compound b16 according to the method described in step A-18 above.

[0169] [B-16 process] Compound B2 can be produced from compound B1 according to the method described in step A-19 above.

[0170] Manufacturing method C Z is a group represented by formula (Z-1), and R1a is a methyl group, and R 1b is a hydrogen atom or a methyl group, and R 2 Ga-(CH2) u In the case of -COOH, the compound represented by formula (1-1) can be produced, for example, by the following production method. [ka] (Wherein, Q, AA, u, b and n are as defined in Section 2, R b and P X is the same as above.)

[0171] Compound c1 represents compound a18 of process A or compound b14 of process B.

[0172] [C-1 process] Compound c2 can be produced from compound c1 according to the method described in step A-16 above.

[0173] [C-2 process] Compound c3 can be produced from compound c2 according to the method described in step A-15 above.

[0174] [C-3 process] Compound c4 can be produced from compound c3 according to the method described in step A-16 above.

[0175] [C-4 process] Compound c5 can be produced from compound c4 according to the method described in step A-17 above.

[0176] [C-5 process] Compound C1 can be produced from compound c5 according to the method described in step A-18 above.

[0177] [C-6 process] Compound C2 can be produced from compound C1 according to the method described in step A-19 above.

[0178] Manufacturing method D Z is a group represented by formula (Z-1), and R 1a is a methyl group, and R 1b is a hydrogen atom or a methyl group, and R 2 Ga-(CH2) u -COR 4 and R 4 Ga-(AB) p When n is 1, 2, 3 or 4, the compound represented by formula (1-1) can be produced, for example, by the following production method. [ka] (Wherein, Q, AA, AB, u, b and p are as defined in item 2, and P Y means a protecting group for an amino group, and R a , R b and P X is the same as above.)

[0179] Above P Y Examples of the protecting group for the amino group represented by the formula (I) include protecting groups described in Protective Groups in Organic Synthesis (Theodora W. Greene, Peter G. M. Wuts, published by John Wiley & Sons, Inc., 1999).

[0180] Compound d1 can be purchased, for example, as a commercial product. Compound d6 represents compound a18 of Production Method A or compound b14 of Production Method B.

[0181] [D-1 process] Compound d2 can be produced by condensing compound d1 with an aspartic acid diester or a glutamic acid diester according to the method described in step A-13 above.

[0182] [D-2 process] Compound d3 can be produced by hydrolyzing the ester of compound d2 according to the method described in step A-12 above.

[0183] [D-3 process] Compound d4 is a mixture of compound d3 and (AB) p can be produced by condensing the above in accordance with the method described in step A-13 above.

[0184] [D-4 process] Compound d5 can be produced by carrying out hydrolysis of the ester of compound d4 and deprotection of the protecting group of the amino group in accordance with the method described in step A-18 above. p When has an ester or protected amino group, hydrolysis of the ester and deprotection of the amino protecting group can be carried out in this deprotection step, if necessary.

[0185] [D-5 process] Compound d7 can be produced from compound d5 and compound d6 according to the method described in step A-16 above.

[0186] [D-6 process] Compound d8 can be produced from compound d7 according to the method described in step A-17 above.

[0187] [D-7 process] Compound D1 can be produced from compound d8 according to the method described in step A-18 above.

[0188] [D-8 process] Compound D2 can be produced from compound D1 according to the method described in step A-19 above.

[0189] Manufacturing method E Z is a group represented by formula (Z-2) or formula (Z-3), and R 1a is a methyl group, and R 1b is a hydrogen atom or a methyl group, and R 3When is -OH, the compound represented by formula (1-1) can be produced, for example, by the following production method. [ka] (In the formula, Q, AA, b, and n are as defined in item 2, g represents 1 or 2, and R b and P X is the same as above.)

[0190] Compound e1 represents compound a18 of process A or compound b14 of process B.

[0191] [E-1 process] Compound e2 can be produced from compound e1 according to the method described in step A-16 above.

[0192] [E-2 process] Compound e3 can be produced from compound e2 according to the method described in step A-17 above.

[0193] [E-3 process] Compound E1 can be produced from compound e3 according to the method described in step A-18 above.

[0194] [E-4 process] Compound E2 can be produced from compound E1 according to the method described in step A-19 above.

[0195] Manufacturing method F Z is a group represented by formula (Z-2) or formula (Z-3), and R 1a is a methyl group, and R 1b is a hydrogen atom or a methyl group, and R 3 When is -OH, the compound represented by formula (1-1) can be produced, for example, by the following production method. [ka] (In the formula, Q, AA, b, and n are as defined in Section 2; g, R b and P X is the same as above.)

[0196] Compound f1 represents compound a18 of production method A or compound b14 of production method B.

[0197] [F-1 process] Compound f2 can be produced from compound f1 according to the method described in step A-16 above.

[0198] [F-2 process] Compound f3 can be produced from compound f2 according to the method described in step A-15 above.

[0199] [F-3 process] Compound f4 can be produced from compound f3 according to the method described in step A-16 above.

[0200] [F-4 process] Compound f5 can be produced from compound f4 according to the method described in step A-17 above.

[0201] [F-5 process] Compound F1 can be produced from compound f5 according to the method described in step A-18 above.

[0202] [F-6 process] Compound F2 can be produced from compound F1 according to the method described in step A-19 above.

[0203] Manufacturing method G Z is a group represented by formula (Z-2) or formula (Z-3), and R 1a is a methyl group, and R 1b is a hydrogen atom or a methyl group, and R 3 Ga-(AB) p In this case, the compound represented by formula (1-1) can be produced, for example, by the following production method. [ka] (In the formula, Q, AA, AB, b, p, and n are as defined in Item 2; g, R a , R b , P X and P Y is the same as above.)

[0204] Compound g1 can be purchased, for example, as a commercial product. Compound g6 represents compound a18 of Production Method A or compound b14 of Production Method B.

[0205] [G-1 process] Compound g2 can be produced by condensing compound g1 with an aspartic acid diester or a glutamic acid diester according to the method described in step A-13 above.

[0206] [G-2 process] Compound g3 can be prepared by hydrolyzing the ester of compound g2 according to the method described in step A-12 above.

[0207] [G-3 process] Compound g4 is the product of compound g3 and (AB) p It can be produced according to the method described in step A-13 above.

[0208] [G-4 process] Compound g5 can be produced by hydrolysis of the ester of compound g4 and deprotection of the amino protecting group in accordance with the method described in step A-18 above. p When has an ester or a protected amino group, hydrolysis of the ester and deprotection of the protecting group of the amino group can be carried out in this deprotection step, if necessary.

[0209] [G-5 process] Compound g7 can be produced from compound g5 and compound g6 according to the method described in step A-16 above.

[0210] [G-6 process] Compound g8 can be produced from compound g7 according to the method described in step A-17 above.

[0211] [G-7 process] Compound G1 can be produced from compound g8 according to the method described in step A-18 above.

[0212] [G-8 process] Compound G2 can be produced from compound G1 according to the method described in step A-19 above.

[0213] Manufacturing method H Z' is a group represented by formula (Z-4), and R 1a is a methyl group, and R 1b When is a hydrogen atom or a methyl group, the compound represented by formula (1-2) can be produced, for example, by the following production method. [ka] (Wherein, Q, AA, h and n are as defined in Section 5, and R x , P X and P Y is the same as above.)

[0214] Compound h1 represents compound a18 of Production Method A or compound b14 of Production Method B. Compound h5 is, for example, commercially available.

[0215] [H-1 process] Compound h2 can be produced from compound h1 according to the method described in step A-16 above.

[0216] [H-2 process] Compound h3 can be produced by condensing compound h2 with a lysine derivative according to the method described in step A-13 above.

[0217] [H-3 process] Compound h4 can be produced from compound h3 according to the method described in step A-18 above.

[0218] [H-4 process] Compound H1 can be produced from compound h4 and compound h5 according to the method described in step A-16 above.

[0219] [H-5 process] Compound H2 can be produced from compound H1 according to the method described in step A-19 above.

[0220] Manufacturing method I Z' is a group represented by formula (Z-5), and R 1a is a methyl group, and R 1b When is a hydrogen atom or methyl, the compound represented by formula (1-2) can be produced, for example, by the following production method. [ka] (Wherein, Q, AA, h and n are as defined in Section 5, and R x , P X and P Y is the same as above.)

[0221] Compound i1 represents compound a18 of Production Method A or compound b14 of Production Method B. Compound i5 is, for example, commercially available.

[0222] [I-1 process] Compound i2 can be produced from compound i1 according to the method described in step A-16 above.

[0223] [I-2 process] Compound i3 can be produced by condensing compound i2 with a lysine derivative according to the method described in step A-13 above.

[0224] [I-3 process] Compound i4 can be produced from compound i3 according to the method described in step A-18 above.

[0225] [I-4 process] Compound I1 can be produced from compound i4 and compound i5 according to the method described in step A-16 above.

[0226] [I-5 process] Compound I2 can be produced from compound I1 according to the method described in step A-19 above.

[0227] Manufacturing method L Compound l6 is a production intermediate of compound L1 represented by formula (1-1), in which Z is a group represented by formula (Z-1), formula (Z-2), or formula (Z-3), and Q is a group represented by formula (Qa-1), formula (Qa-2), formula (Qa-3), formula (Qa-4), formula (Qa-5), or formula (Qa-6). Compound l6 can be produced, for example, by the following production method. Compound L1 can also be produced from compound l6 according to the production method described in steps A-16 to A-19 of production method A. [ka] (In the formula, R 1a and b is as defined in Item 1, R a is C 1-6 represents an alkyl group.

[0228] Compound l1 can be purchased as a commercial product, for example. Compound a15 can be produced, for example, by the method described in Production Method A.

[0229] [L-1 process] Compound 12 can be produced, for example, according to the method described in Bioorg. Med. Chem. Lett. 14 (2004) 5317-5322.

[0230] [L-2 process] Compound l3 can be produced, for example, according to the method described in Bioorg. Med. Chem. Lett. 14 (2004) 5317-5322 and the like.

[0231] [L-3 process] Compound 14 can be produced, for example, according to the method described in Bioorg. Med. Chem. Lett. 14 (2004) 5317-5322.

[0232] [L-4 process] Compound l5 can be produced from compound l4 according to step A-14 of production method A.

[0233] [L-5 process] Compound l6 can be produced from compound l5 according to step A-15 of production method A.

[0234] Manufacturing method M Compound m7 is a production intermediate of compound Ma1 represented by formula (1-1), in which Z is a group represented by formula (Z-1), formula (Z-2), or formula (Z-3), and Q is a group represented by formula (Qa-7). Compound m7 can be produced, for example, by the following production method. Compound Ma1 can be produced from compound m7 in accordance with the production method described in steps A-16 to A-19 of production method A. [ka] (In the formula, R 1a and b is as defined in Item 1, R a is C 1-6 represents an alkyl group.

[0235] Compound m1 can be purchased as a commercial product, for example. Compound a15 can be produced, for example, by the method described in Production Method A.

[0236] [M-1 process] Compound m2 can be produced, for example, according to the method described in Bioorg. Med. Chem. Lett. 14 (2004) 5317-5322 or the like.

[0237] [M-2 process] Compound m3 can be produced, for example, according to the method described in Bioorg. Med. Chem. Lett. 14 (2004) 5317-5322 and the like.

[0238] [M-3 process] Compound m4 can be produced, for example, according to the method described in J. Med. Chem. 2004, 47, 4774-4786.

[0239] [M-4 process] Compound m5 can be produced, for example, according to the method described in Bioorg. Med. Chem. Lett. 14 (2004) 5317-5322 and the like.

[0240] [M-5 process] Compound m6 can be produced from compound m5 according to step A-14 of Production Method A.

[0241] [M-6 process] Compound m7 can be produced from compound m6 according to step A-15 of Production Method A.

[0242] Manufacturing method N Compound n5 is a production intermediate of compound N1 represented by formula (1-1), in which Z is a group represented by formula (Za-1), formula (Za-2) or formula (Za-3). Compound n5 can be produced, for example, by the following production method. Compound N1 can also be produced from compound n5 in accordance with the production method described in steps A-16 to A-18 of production method A. [ka] (In the formula, R aa and b is as defined in Item 1, R a is C 1-6 represents an alkyl group.

[0243] Compound n1 can be purchased as a commercial product, for example. Compound a15 can be produced, for example, by the method described in Production Method A.

[0244] [N-1 process] Compound n2 can be produced, for example, according to the method described in WO 2003 / 082268.

[0245] [N-2 process] Compound n3 can be produced from compound n2 according to the method described in step A-13.

[0246] [N-3 process] Compound n4 can be produced from compound n3 according to the method described in step A-14.

[0247] [N-4 process] Compound n5 can be produced from compound n4 according to step A-15 of production method A.

[0248] Manufacturing method O Compound O1 and compound O2 are compounds represented by formula (1-1) in which Z is a group represented by formula (Za-4) or formula (Za-5). Compound O1 and compound O2 can be produced, for example, by the following production method. [ka] (In the formula, AA, R 1a , b and n are as defined in item 1, EL represents a chlorine atom, a bromine atom, an iodine atom, a trifluoromethylsulfonyl group or a tosyl group, R zz is a hydrogen atom or -COOR a represents P x represents a protecting group for an amino group, Pz represents a protecting group for a carboxyl group, and R a is C 1-6 represents an alkyl group.

[0249] Compound o1 can be prepared by the method described in, for example, WO 2004 / 026293, WO 2016 / 123582, etc., or can be purchased commercially. Compound a15 can be prepared by the method described in Production Method A, for example.

[0250] [O-1 process] Compound o2 can be produced from compound o1 according to the methods described in, for example, WO 2004 / 026293, Bioorg. Med. Chem. Lett. 14 (2004) 5317-5322, and the like.

[0251] [O-2 process] Compound o3 can be produced from compound o2 according to the method described in step A-8 above.

[0252] [O-3 process] Compound o4 can be produced from compound o3 according to the method described in step A-11 above.

[0253] [O-4 process] Compound o5 can be produced from compound o4 by, for example, the method described in WO 2004 / 026293.

[0254] [O-5 process] Compound o6 can be produced from compound o5 by the method described in, for example, WO 2004 / 026293.

[0255] [O-6 process] Compound o7 can be produced from compound o6 by, for example, the method described in International Publication No. 2004 / 026293.

[0256] [O-7 process] Compound o8 is prepared by converting the carboxyl group of compound o7 into a protecting group P Z This step can be carried out in accordance with the method described in Protective Groups in Organic Synthesis (Theodora W. Greene, Peter GM Wuts, John Wiley & Sons, Inc., 1999).

[0257] [O-8 process] Compound o9 can be produced from compound o8 according to the method described in step A-12 above.

[0258] [O-9 process] Compound o10 can be produced from compound o9 according to the method described in step A-13 above.

[0259] [O-10 process] Compound o11 can be produced from compound o10 according to the method described in step A-14 above.

[0260] [O-11 process] Compound o12 can be produced from compound o11 according to the method described in step A-15 above.

[0261] [O-12 process] Compound o13 can be produced from compound o12 according to the method described in step A-16 above.

[0262] [O-13 process] Compound o14 can be prepared by removing the protecting group P from compound o13. z This step can be carried out in accordance with the method described in Protective Groups in Organic Synthesis (Theodora W. Greene, Peter GM Wuts, John Wiley & Sons, Inc., 1999), or the like.

[0263] [O-14 process] Compound O1 can be produced from compound O14 according to the method described in step A-17 above.

[0264] [O-15 process] Compound O2 can be produced from compound O1 according to the method described in step A-19 above.

[0265] Manufacturing method P Compound P1 and compound P2 are compounds represented by formula (1-2) in which Z is a group represented by formula (Za-8) or formula (Za-9). Compound P1 and compound P2 can be produced, for example, by the following production method. [ka] (Wherein, AA, n, h and R 1a is as defined in Section 4, and P x represents a protecting group for an amino group, P z represents a protecting group for a hydroxyl group, and R a is C 1-6 represents an alkyl group.

[0266] Compound p1 can be purchased as a commercial product, for example. Compound a15 can be produced, for example, by the method described in Production Method A.

[0267] [P-1 process] Compound p2 can be produced from compound p1 according to the method described in, for example, WO 2004 / 026293.

[0268] [P-2 process] Compound p3 is obtained by removing the protecting group P from compound p2. zThis step can be carried out in accordance with the method described in Protective Groups in Organic Synthesis (Theodora W. Greene, Peter GM Wuts, John Wiley & Sons, Inc., 1999), or the like.

[0269] [P-3 process] Compound p4 can be produced from compound p3 according to the method described in, for example, WO 2004 / 026293.

[0270] [P-4 process] Compound p5 is prepared by converting the amino group and hydroxyl group of compound p4 into a protecting group P x and a protecting group P z This step can be carried out in accordance with the method described in Protective Groups in Organic Synthesis (Theodora W. Greene, Peter GM Wuts, John Wiley & Sons, Inc., 1999).

[0271] [P-5 process] Compound p6 can be prepared by removing the protecting group P from compound p5. z This step can be carried out in accordance with the method described in Protective Groups in Organic Synthesis (Theodora W. Greene, Peter GM Wuts, John Wiley & Sons, Inc., 1999) or Tetrahedron Lett. 45 (2004) 495-499.

[0272] [P-6 process] Compound p7 can be produced from compound p6 according to the method described in step A-13.

[0273] [P-7 process] Compound p8 can be produced from compound p7 according to the method described in step A-18 above.

[0274] [P-8 process] Compound p9 can be produced from compound p8 by condensation according to the method described in step A-13 above. n In the case where has an ester or a protected amino group, after the condensation reaction, hydrolysis of the ester and deprotection of the protecting group of the amino group can be carried out, if necessary, in accordance with the method described in Protective Groups in Organic Synthesis (Theodora W. Greene, Peter GM Wuts, published by John Wiley & Sons, Inc., 1999).

[0275] [P-9 process] Compound P1 can be produced by condensing compound p9 with a succinimide derivative according to the method described in step A-16 above.

[0276] [P-10 process] Compound P2 can be produced from compound P1 according to the method described in step A-19 above.

[0277] Manufacturing method T Compound T1 and Compound T2 are compounds represented by formula (1-2) in which Z is a group represented by formula (Za-6) or formula (Za-7). Compound T1 and Compound T2 can be produced, for example, by the following production method. [ka] (Wherein, AA, n, h and R 1a is as defined in item 4, EL represents a chlorine atom, a bromine atom, an iodine atom, a trifluoromethylsulfonyl group, or a tosyl group, P x and P y represents a protecting group for an amino group, and R a is C 1-6represents an alkyl group.

[0278] Compound t1 can be prepared by the method described in, for example, WO 2004 / 026293, WO 2016 / 123582, etc., or can be purchased as a commercial product. Compound a15 can be prepared by, for example, the method described in Production Method A.

[0279] [T-1 process] Compound t2 can be produced from compound t1 according to the methods described in, for example, International Publication No. 2004 / 026293, Bioorg. Med. Chem. Lett. 14 (2004) 5317-5322, and the like.

[0280] [T-2 process] Compound t3 can be produced from compound t2 according to the method described in step A-8 above.

[0281] [T-3 process] Compound t4 can be produced from compound t3 by, for example, the method described in International Publication No. 2016 / 123582.

[0282] [T-4 process] Compound t5 is a compound t4 having an amino group protected by a protecting group P y This step can be carried out in accordance with the method described in Protective Groups in Organic Synthesis (Theodora W. Greene, Peter GM Wuts, John Wiley & Sons, Inc., 1999).

[0283] [T-5 process] Compound t6 can be produced from compound t5 according to the method described in the above step A-13.

[0284] [T-6 process] Compound t7 can be produced from compound t6 according to the method described in step A-14 above.

[0285] [T-7 process] Compound t8 can be produced from compound t7 according to the method described in the above step A-13.

[0286] [T-8 process] Compound t9 is a compound having a protecting group P x and P y This step can be carried out in accordance with the method described in Protective Groups in Organic Synthesis (Theodora W. Greene, Peter GM Wuts, John Wiley & Sons, Inc., 1999), Tetrahedron Lett. 45 (2004) 495-499, etc. n When has an ester or a protected amino group, hydrolysis of the ester and deprotection of the protecting group of the amino group can be carried out in this deprotection step, if necessary.

[0287] [T-9 process] Compound T1 can be produced from compound t9 according to the method described in the above step A-13 or A-16.

[0288] [T-10 process] Compound T2 can be produced from compound T2 according to the method described in step A-19 above.

[0289] Manufacturing method U Compound U1 and compound U2 are compounds represented by formula (1-2) in which Z is a group represented by formula (Za-10). Compound U1 and compound U2 can be produced, for example, by the following production method. [ka] (Wherein, AA, h, Q, n and R 1a is as defined in Section 4, and P xrepresents a protecting group for an amino group.

[0290] [U-1 process] Compound U1 can be produced from compound c2 according to the method described in A-18 above.

[0291] [U-2 process] Compound U2 can be produced from compound U1 according to the method described in A-13 or A-16 above.

[0292] Manufacturing method W Compound W1 is a compound represented by formula (1-2) in which Z is a group represented by formula (Za-11). Compound W1 can be produced, for example, by the following production method. [ka] (Wherein, AA, h and n are as defined in Section 4, P x represents a protecting group for an amino group, and R a is C 1-6 represents an alkyl group.

[0293] Compound w1 can be purchased as a commercial product, for example. Compound a15 can be produced, for example, by the method described in Production Method A.

[0294] [W-1 process] Compound w2 is prepared by converting the amino group of compound w1 into a protecting group P x This step can be carried out in accordance with the method described in Protective Groups in Organic Synthesis (Theodora W. Greene, Peter GM Wuts, John Wiley & Sons, Inc., 1999).

[0295] [W-2 process] Compound w3 can be produced from compound w2 according to the method described in the above step A-13.

[0296] [W-3 process] Compound w4 can be produced from compound w3 according to the method described in the above step A-14.

[0297] [W-4 process] Compound w5 can be produced from compound w4 according to the method described in the above step A-13.

[0298] [W-5 process] Compound w6 is a compound obtained by removing the protecting group P x This step can be carried out in accordance with the method described in Protective Groups in Organic Synthesis (Theodora W. Greene, Peter GM Wuts, John Wiley & Sons, Inc., 1999), Tetrahedron Lett. 45 (2004) 495-499, etc. n When has an ester or a protected amino group, hydrolysis of the ester and deprotection of the protecting group of the amino group can be carried out in this deprotection step, if necessary.

[0299] [W-6 process] Compound W1 can be produced from compound w6 according to the method described in step A-13 or A-16 above.

[0300] The method for producing the antibody-drug conjugate of the present invention is described below. The antibody-drug conjugate of the present invention represented by formula (2-1) or (2-2) can be produced, for example, by the following production method J or production method K.

[0301] Manufacturing method J [ka] (In the formula, mAb, q, b, and Z are as defined in item 16, mAb' represents a mAb whose disulfide bond has been reduced, and qq represents an integer of 1 to 8.)

[0302] [J-1 process] Compound j2 can be produced by reacting compound j1 with an appropriate disulfide reducing agent in an appropriate buffer solution. Examples of disulfide reducing agents include dithiothreitol, mercaptoethanol, tris(2-carboxyethyl)phosphine, and the like, preferably tris(2-carboxyethyl)phosphine. Examples of buffer solutions include Tris-HCl, PBS, HEPES, acetate-based, borate-based, phosphate-based, and carbonate-based buffer solutions, preferably Tris-HCl and PBS. The pH during the reaction is usually 2 to 12, and preferably 4 to 9. The reaction time is usually 5 minutes to 24 hours, and preferably 5 minutes to 5 hours. The reaction temperature is usually -10°C to 50°C, and preferably 0°C to 40°C.

[0303] [J-2 process] Compound J1 can be produced by reacting compound j2 with compound j3 in an appropriate buffer solution. Examples of the buffer solution include Tris-HCl, PBS, HEPES, acetate, borate, phosphate, and carbonate buffer solutions, and preferably Tris-HCl and PBS. The pH during the reaction is usually 2 to 12, and preferably 4 to 9. The reaction time is usually 5 minutes to 72 hours, and preferably 30 minutes to 24 hours. The reaction temperature is usually -78°C to 200°C, and preferably 0°C to 25°C.

[0304] Manufacturing method K [ka] (In the formula, mAb, q, h, and Z' are as defined in item 19, mAb' represents a mAb whose disulfide bond has been reduced, and qq represents an integer of 1 to 8.)

[0305] [K-1 process] Compound k2 can be produced from compound k1 according to the method described in step J-1 above.

[0306] [K-2 process] Compound K1 can be produced from compound k2 and compound k3 according to the method described in step J-2 above.

[0307] The methods for producing the hemiasterlin derivative and antibody-drug conjugate of the present invention are described above. However, the hemiasterlin derivative and antibody-drug conjugate of the present invention can also be produced by other methods, for example, by appropriately combining methods known to those skilled in the art.

[0308] The appropriate base used in each step of each of the above-mentioned production methods should be appropriately selected depending on the type of reaction or raw material compound, and examples thereof include alkali bicarbonates such as sodium bicarbonate and potassium bicarbonate; alkali carbonates such as sodium carbonate and potassium carbonate; metal hydrides such as sodium hydride and potassium hydride; alkali metal hydroxides such as sodium hydroxide and potassium hydroxide; alkali metal alkoxides such as sodium methoxide and sodium t-butoxide; organometallic bases such as butyllithium and lithium diisopropylamide; and organic bases such as triethylamine, diisopropylethylamine, pyridine, 4-dimethylaminopyridine (DMAP), and 1,8-diazabicyclo[5.4.0]-7-undecene (DBU).

[0309] The appropriate solvent used in each step of the above-mentioned production methods should be selected appropriately depending on the type of reaction and raw material compound, and examples of the solvent include alcohols such as methanol, ethanol, isopropanol, etc.; ketones such as acetone, methyl ketone, etc.; halogenated hydrocarbons such as methylene chloride, chloroform, etc.; ethers such as tetrahydrofuran (THF), dioxane, etc.; aromatic hydrocarbons such as toluene, benzene, etc.; aliphatic hydrocarbons such as hexane, heptane, etc.; esters such as ethyl acetate, propyl acetate, etc.; amides such as N,N-dimethylformamide (DMF), N-methyl-2-pyrrolidone, etc.; sulfoxides such as dimethyl sulfoxide (DMSO), etc.; nitriles such as acetonitrile, etc.; distilled water, etc., and these solvents can be used alone or in combination of two or more. Depending on the type of reaction, organic bases such as triethylamine, diisopropylethylamine, pyridine, etc. may be used as a solvent.

[0310] The hemiasterlin derivative and antibody-drug conjugate of the present invention can be separated and purified by methods known to those skilled in the art, such as extraction, distribution, reprecipitation, column chromatography (e.g., silica gel column chromatography, ion exchange column chromatography, or preparative liquid chromatography), or recrystallization.

[0311] Examples of recrystallization solvents that can be used include alcohol-based solvents such as methanol, ethanol, and 2-propanol; ether-based solvents such as diethyl ether; ester-based solvents such as ethyl acetate; aromatic hydrocarbon-based solvents such as benzene and toluene; ketone-based solvents such as acetone; halogen-based solvents such as dichloromethane and chloroform; hydrocarbon-based solvents such as hexane; aprotic solvents such as dimethylformamide, acetonitrile; water; or a mixture of these solvents.

[0312] Other purification methods that can be used include the methods described in Experimental Chemistry Lectures (edited by the Chemical Society of Japan, Maruzen), Vol. 1, etc. In addition, the molecular structures of the hemiasterlin derivative and antibody-drug conjugate of the present invention can be easily determined by spectroscopic methods such as nuclear magnetic resonance, infrared absorption, and circular dichroism spectroscopy, or mass spectrometry, with reference to the structures derived from the respective raw material compounds.

[0313] In addition, the intermediate or final product in the above-mentioned production method can be led to another compound included in the present invention by appropriately converting its functional group, particularly by extending various side chains using amino groups, hydroxyl groups, carbonyl groups, halogen atoms, etc. as a foothold, and by protecting and deprotecting the functional groups as necessary. The conversion of functional groups and the extension of side chains can be carried out by a commonly used general method (see, for example, Comprehensive Organic Transformations, RC Larock, John Wiley & Sons Inc. (1999), etc.).

[0314] The hemiasterlin derivative and antibody-drug conjugate of the present invention may have asymmetry or a substituent with an asymmetric carbon, and such compounds have optical isomers. Optical isomers can be produced according to a conventional method. Examples of the production method include a method using a raw material with an asymmetric center or a method of introducing asymmetry at an intermediate stage. For example, in the case of optical isomers, optical isomers can be obtained by using optically active raw materials or by performing optical resolution at an appropriate stage of the production process. As an optical resolution method, for example, when the hemiasterlin derivative and antibody-drug conjugate of the present invention have a basic functional group, an optically active acid (e.g., monocarboxylic acid such as mandelic acid, N-benzyloxyalanine, lactic acid, etc.; dicarboxylic acid such as tartaric acid, o-diisopropylidene tartaric acid, malic acid, etc.; sulfonic acid such as camphorsulfonic acid, bromocamphorsulfonic acid, etc.) is used in an inert solvent (e.g., alcoholic solvents such as methanol, ethanol, 2-propanol, etc.; ether solvents such as diethyl ether; ester solvents such as ethyl acetate; hydrocarbon solvents such as toluene; aprotic solvents such as acetonitrile; or a mixed solvent of two or more of the above solvents) is used to form a salt, and a diastereomeric method is included. When the hemiasterlin derivative of the present invention or a synthetic intermediate thereof has an acidic functional group such as carboxyl, optical resolution can also be performed by forming a salt using an optically active amine (e.g., organic amines such as 1-phenylethylamine, quinine, quinidine, cinchonidine, cinchonine, strychnine, etc.).

[0315] The temperature at which the salt is formed may range from -50°C to the boiling point of the solvent, preferably from 0°C to the boiling point, more preferably from room temperature to the boiling point of the solvent. In order to improve the optical purity, it is desirable to once raise the temperature to near the boiling point of the solvent. When filtering out the precipitated salt, the yield can be improved by cooling as necessary. The amount of the optically active acid or amine used may range from about 0.5 to about 2.0 equivalents relative to the substrate, preferably about 1 equivalent. If necessary, the crystals may be recrystallized in an inert solvent (e.g., alcohol-based solvents such as methanol, ethanol, and 2-propanol; ether-based solvents such as diethyl ether; ester-based solvents such as ethyl acetate; hydrocarbon-based solvents such as toluene; aprotic solvents such as acetonitrile; or a mixed solvent of two or more of the above solvents) to obtain a high-purity optically active salt. If necessary, the optically resolved salt may be treated with an acid or base by a conventional method to obtain a free form.

[0316] Among the raw materials or intermediates in each of the above-mentioned production methods, those for which the production method is not particularly described are commercially available compounds or can be synthesized from commercially available compounds by methods known to those skilled in the art or methods similar thereto.

[0317] The antibody-drug conjugate of the present invention and a pharmaceutical composition comprising the same are useful as anti-cancer agents (e.g., therapeutic agents for breast cancer, gastric cancer, lung cancer, liver cancer, cervical cancer, colorectal cancer, rectal cancer, colon cancer, glioma, lymphoma, ovarian cancer, pancreatic cancer, prostate cancer, kidney cancer, urothelial cancer, skin cancer, thyroid cancer, bladder cancer, head and neck cancer, uterine cancer, mesothelioma, melanoma, multiple myeloma, leukemia, etc.).

[0318] The antibody-drug conjugate of the present invention can be administered orally or parenterally, either directly or after preparation using an appropriate dosage form. Examples of dosage forms include, but are not limited to, solutions, suspensions, injections, etc. These preparations are produced by known methods using pharma- ceutically acceptable additives.

[0319] As additives, depending on the purpose, excipients, disintegrants, binders, fluidizing agents, lubricants, coating agents, solubilizers, solubilizing aids, thickeners, dispersants, stabilizers, sweeteners, flavors, etc. can be used. Specific examples include lactose, mannitol, crystalline cellulose, low-substituted hydroxypropyl cellulose, corn starch, partially pregelatinized starch, carmellose calcium, croscarmellose sodium, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, polyvinyl alcohol, magnesium stearate, sodium stearyl fumarate, polyethylene glycol, propylene glycol, titanium oxide, talc, etc.

[0320] Examples of the "anticancer compounds" that can be used in conjunction or in combination with the antibody-drug conjugate of the present invention include at least one or more antitumor agents selected from the group consisting of anticancer alkylating agents, anticancer antimetabolites, anticancer antibiotics, anticancer platinum coordination compounds, anticancer camptothecin derivatives, anticancer tyrosine kinase inhibitors, anticancer serine threonine kinase inhibitors, anticancer phospholipid kinase inhibitors, anticancer monoclonal antibodies, interferons, biological response modifiers, hormones, immune checkpoint inhibitors, epigenetics-related molecule inhibitors, protein post-translational modification inhibitors, and other antitumor agents. Specific examples of "anticancer compounds" that can be used in combination or in combination with other compounds include, for example, azacitidine, vorinostat, decitabine, romidepsin, idarubicin, daunorubicin, doxorubicin, enocitabine, cytarabine, mitoxantrone, thioguanine, etoposide, ifosfamide, cyclophosphamide, dacarbazine, temozolomide, nimustine, busulfan, procarbazine, melphalan, ranimustine, all-trans retinoic acid, tamibarotene, cisplatin, carboplatin, oxaliplatin, irinotecan, bleomycin, mitomycin C, methotrexate, paclitaxel, docetaxel, gemcitabine, tamoxifen, thiotepa, tegafur, fluorouracil, and the like. Rasil, everolimus, temsirolimus, gefitinib, erlotinib, imatinib, crizotinib, osimertinib, afatinib, dasatinib, bosutinib, vandetanib, sunitinib, axitinib, pazopanib, lenvatinib, lapatinib, nilotinib, ibrutinib, ceritinib, alectinib, tofacitinib, baricitinib, ruxolitinib These include nib, olaparib, sorafenib, vemurafenib, dabrafenib, trametinib, palbociclib, bortezomib, carfilzomib, rituximab, cetuximab, trastuzumab, bevacizumab, panitumumab, nivolumab, atezolizumab, mogamulizumab, alemtuzumab, ofatumumab, ipilimumab, ramucirumab, brentuximab vedotin, trastuzumab emtansine, gemtuzumab ozogamicin, and inotuzumab ozogamicin.

[0321] As described above, the antibody-drug conjugate of the present invention and the pharmaceutical composition containing the same can be used for the treatment of cancer. That is, one aspect of the present invention can be said to be a method for treating cancer, which comprises administering the antibody-drug conjugate or the pharmaceutical composition containing the same to a subject suffering from cancer. The subject suffering from cancer may be a human patient or a non-human animal. EXAMPLES

[0322] The present invention will be described in more detail below with reference examples, examples and test examples, but the present invention is not limited thereto. Note that the compound names shown in the following reference examples and examples do not necessarily conform to the IUPAC nomenclature.

[0323] The compounds of the Reference Examples and Examples may be obtained as acid addition salts such as TFA salts by post-reaction treatment or other methods.

[0324] In order to simplify the description in the specification, the following abbreviations may be used in the Reference Examples, Examples, and Tables in the Examples. The abbreviations used as substituents are Me, Et, Boc, tert-butoxycarbonyl, Fmoc, 9-fluorenylmethyloxycarbonyl, trt, trityl, Ph, and Bn, respectively. TFA means trifluoroacetic acid, THF means tetrahydrofuran, TCEP means tris(2-carboxyethyl)phosphine, Tris-HCl means trishydroxymethylaminomethane hydrochloride, PBS means phosphate buffered saline, HEPES means 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid, and PIPES means piperazine-1,4-bis(2-ethanesulfonic acid). The symbols used in NMR are s for singlet, d for doublet, dd for doublet of doublets, t for triplet, q for quartet, m for multiplet, br for broad, brs for broad singlet, brd for broad doublet, brm for broad multiplet, and J for coupling constant.

[0325] The measurement conditions for the high-performance liquid chromatography mass spectrometer (LCMS) were as follows, and the observed mass spectrometry values ​​[MS (m / z)] were defined as [M+nH] n+ / n, [M+Na] + , [M-nH] n- / n, and retention time is Rt(min). For each measured value, the measurement conditions used are indicated as A to D or F to H.

[0326] Measurement condition A Detection equipment: Shimadzu LCMS-IT-TOF Column: Phenomenex Kinetex(1.7μm C18,50mm×2.10mm) Solvent: A liquid: 0.1% HCOOH / CH3CN, B liquid: 0.1% HCOOH / H2O Gradient Condition: 0.0 minutes;A / B=1:99 0.0-1.4min Linear gradient from A 1% to 95% 1.4-1.6 minutes; A / B=95:5 1.6-2.0 minutes;A / B=1:99 Flow rate: 1.2mL / min UV: 220 / 254nm Column temperature: 40℃

[0327] Measurement condition B Detection equipment: Shimadzu LCMS-IT-TOF Column: Phenomenex Kinetex(1.7μm C18,50mm×2.10mm) Solvent: A liquid: 0.1% HCOOH / CH3CN, B liquid: 0.1% HCOOH / H2O Gradient Condition: 0.0 minutes;A / B=10:90 0.0-1.4min Linear gradient from A 10% to 90% 1.4-1.6 minutes; A / B=90:10 1.6-2.0 points; A / B=10:90 Flow rate:1.2mL / min UV:220 / 254nm カラムTemperature:40℃

[0328] Measurement conditions C Detection equipment: Shimadzu LCMS-IT-TOF Column:Phenomenex Kinetex(1.7μm C8,50mm×2.10mm) Solvent: Solution A: 0.1% HCOOH / CH3CN, Solution B: 0.1% HCOOH / H2O Gradient Condition: 0.0 points; A / B=1:99 0.0-1.4 points Linear gradient from A 1% to 95% 1.4-1.6 points; A / B=95:5 1.6-2.0 points; A / B=1:99 Flow rate:1.2mL / min UV:220 / 254nm カラムTemperature:40℃

[0329] Measurement condition D Detection equipment: Shimadzu LCMS-IT-TOF Column:Phenomenex Kinetex(1.7μm C8,50mm×2.10mm) Solvent: Solution A: 0.1% HCOOH / CH3CN, Solution B: 0.1% HCOOH / H2O Gradient Condition: 0.0 points; A / B=10:90 0.0-1.4 points Linear gradient from A 10% to 90% 1.4-1.6 points; A / B=90:10 1.6-2.0 points; A / B=10:90 Flow rate:1.2mL / min UV:220 / 254nm Column temperature: 40℃

[0330] Measurement condition F Detection equipment: ACQUITY (registered trademark) SQdetector (Waters Corporation) HPLC: ACQUITY® system Column: Waters ACQUITY UPLC (registered trademark) BEH C18 (1.7 μm, 2.1 mm × 30 mm) Solvent: Solution A: 0.06% formic acid / CH3CN, Solution B: 0.06% formic acid / H2O Gradient Condition:0.0-1.3min Linear gradient from A 2% to 96% Flow rate: 0.8mL / min UV: 220 / 254nm Column temperature: 25℃

[0331] Measurement condition G Detection equipment: Shimadzu LCMS-IT-TOF Column: Phenomenex Kinetex(1.7μm C8,50mm×2.10mm) Solvent: A liquid: 0.1% HCOOH / CH3CN, B liquid: 0.1% HCOOH / H2O Gradient Condition: 0.0 minutes;A / B=10:90 0.0-1.4min Linear gradient from A 10% to 95% 1.4-1.6 minutes; A / B=95:5 1.6-2.0 minutes;A / B=10:90 Flow rate: 1.2mL / min UV: 220 / 254nm Column temperature: 40℃

[0332] Measurement condition H Detection equipment: Shimadzu LCMS-IT-TOF Column: Phenomenex Kinetex(1.7μm C8,50mm×2.10mm) Solvent: A liquid: 0.1% HCOOH / CH3CN, B liquid: 0.1% HCOOH / H2O Gradient Condition: 0.0 minutes;A / B=40:60 0.0-1.4min Linear gradient from A 40% to 95% 1.4-1.6 minutes; A / B=95:5 1.6-2.0 minutes;A / B=5:95 Flow rate: 1.2mL / min UV: 220 / 254nm Column temperature: 40℃

[0333] High performance liquid chromatograph: The measurement conditions for determining the average drug-antibody ratio (average DAR) are as follows, and the retention time is indicated as Rt (min). For each measured value, the measurement conditions used for the measurement are indicated with E or I.

[0334] Measurement condition E HPLC: Shimadzu LC-10A series Column:nonporous TSKgel Butyl-NPR column(TosohBioscience,2.5μm,35mm×4.6mm) Solvent: Solution A: 1.5 mol / L ammonium sulfate, 25 mmol / L sodium phosphate aqueous solution (pH 6.95), Solution B: 25% isopropanol / 25 mmol / L sodium phosphate aqueous solution (pH 6.95) Gradient Condition: 0.0 minutes;A / B=100:0 0.0-12.0min Linear gradient from B 0% to 100% 12.1-18.0 minutes;A / B=100:0 Flow rate: 0.8mL / min UV: 230nm Column temperature: 25℃

[0335] Measurement condition I HPLC: Shimadzu LC-10A series Column:nonporous TSKgel Butyl-NPR column (TosohBioscience, 2.5μm, 35mm×4.6mm) Solvent: Solution A: 1.5 mol / L ammonium sulfate, 25 mmol / L sodium phosphate aqueous solution (pH 6.95), Solution B: 25% isopropanol / 25 mmol / L sodium phosphate aqueous solution (pH 6.95) Gradient Condition: 0.0 minutes;A / B=100:0 0.0-24.0min Linear gradient from B 0% to 100% 24.1-30.0 minutes;A / B=100:0 Flow rate: 0.8mL / min UV: 230nm Column temperature: 25℃

[0336] Reference example 1 tert-Butyl (6S,9S,12S,13E,17R)-9-tert-butyl-17-{3-[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]amino}-3-oxopropyl)-2,2,5,11,14-pentamethyl-6-[2-(1-methyl-1H-indol-3-yl)propan-2-yl]-4,7,10,15-tetraoxo-12-(propan-2-yl)-3-oxo-5,8,11,16-tetraazaoctadec-13-ene-18-carboxylate [ka]

[0337] a) Preparation of methyl 2-(1-methyl-1H-indol-3-yl)propanoate (compound A1) Under a nitrogen atmosphere, potassium hexamethyldisilazide (1 mol / L tetrahydrofuran solution, 65.5 mL) was added dropwise to a solution of indole-3-acetic acid methyl ester (3.8 g) in tetrahydrofuran (87 mL) at -78 ° C, and the mixture was stirred at 0 ° C for 2 hours. The reaction solution was cooled to -78 ° C, and methyl iodide (23 g) was added dropwise, and the mixture was stirred at 0 ° C for 3 hours. After the reaction was completed, water was added and the mixture was extracted with diethyl ether. The organic layer was washed with saturated saline, dried over anhydrous magnesium sulfate, and the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (elution solvent: hexane: ethyl acetate) to obtain compound A1 (3.95 g). 1 H-NMR(400 MHz, CDCl3):1.60(3H, d, J =7.1 Hz),3.67 (3H, s), 3.76 (3H, s), 4.02 (1H, q, J = 7.1 Hz), 7.00(1H, s),7.12 (1H, t,J = 7.8 Hz), 7.23 (1H, t,J = 7.8 Hz), 7.29 (1H, d,J = 7.8 Hz),7.66(1H, d, J= 7.8Hz).

[0338] b) Preparation of methyl 2-methyl-2-(1-methyl-1H-indol-3-yl)propanoate (compound A2) Under a nitrogen atmosphere, potassium hexamethyldisilazide (1 mol / L tetrahydrofuran solution, 27.7 mL) was added dropwise to a solution of compound A1 (3.94 g) in tetrahydrofuran (200 mL) at -78 ° C, and the mixture was stirred at 0 ° C for 2 hours. The reaction solution was cooled to -78 ° C, and methyl iodide (15.4 g) was added dropwise, and the mixture was stirred at 0 ° C for 3 hours. After the reaction was completed, water was added and the mixture was extracted with diethyl ether. The organic layer was washed with saturated saline, dried over anhydrous magnesium sulfate, and the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (elution solvent: hexane: ethyl acetate) to obtain compound A2 (3.59 g). 1H-NMR(400 MHz, CDCl3):1.66(6H,s), 3.61 (3H,s), 3.73 (3H,s), 6.91 (1H,s), 7.06 (1H, t,J = 8.0 Hz), 7.19(1H, t,J = 8.0 Hz), 7.27 (1H,d,J = 8.0 Hz), 7.61 (1H, d,J =7.9 Hz).

[0339] c) Preparation of 2-methyl-2-(1-methyl-1H-indol-3-yl)propan-1-ol (compound A3) Under a nitrogen atmosphere, diisobutylaluminum hydride (1 mol / L n-hexane solution, 38.8 mL) was added dropwise to a solution of compound A2 (3.59 g) in diethyl ether (169 mL) and dichloromethane (47 mL) at -78 ° C, and then the mixture was stirred at 0 ° C for 1 hour. After the reaction was completed, water was added, and the reaction mixture at 25 ° C was added with a saturated aqueous solution of potassium sodium tartrate, followed by extraction with diethyl ether. The organic layer was washed with saturated saline, dried over anhydrous magnesium sulfate, and the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (elution solvent: hexane: ethyl acetate) to obtain compound A3 (3.14 g). 1 H-NMR(400 MHz, CDCl3):1.42(6H,s), 3.74 (3H,s), 3.77 (2H,s), 6.87 (1H,s), 7.07 (1H,t, J = 7.9 Hz), 7.20(1H,t, J = 7.9 Hz), 7.29(1H,d, J = 8.0 Hz), 7.75 (1H,d,J = 8.0 Hz).

[0340] d) Preparation of 2-methyl-2-(1-methyl-1H-indol-3-yl)propanal (Compound A4) Under a nitrogen atmosphere, a mixture of compound A3 (3.14 g), tetrapropylammonium perruthenate (271 mg), N-methylmorpholine-N-oxide (3.26 g) and molecular sieve 4A (7.7 g) in dichloromethane (110 mL) was stirred for 1 hour at 25° C. After the reaction was completed, the mixture was filtered through Celite, the solvent was distilled off, and the residue was purified by silica gel column chromatography (elution solvent: hexane:ethyl acetate) to obtain compound A4 (2.4 g). 1 H-NMR(400 MHz, CDCl3):1.53(6H,s), 3.77 (3H,s), 6.94 (1H,s), 7.07 (1H,t, J = 8.0 Hz), 7.22 (1H,t, J = 8.0Hz), 7.30 (1H,d, J = 8.0 Hz), 7.53(1H,d, J = 8.0 Hz), 9.47 (1H,s).

[0341] e) Preparation of (2S)-2-{[(1R)-2-hydroxy-1-phenylethyl]amino}-3-methyl-3-(1-methyl-1H-indol-3-yl)butanenitrile (Compound A5) Under a nitrogen atmosphere, a solution of compound A4 (2.4 g) and (R)-(-)-2-phenylglycinol (1.63 g) in toluene (47 mL) was heated to reflux for 1.5 hours, water was removed using a Dean-Stark apparatus, and the solvent was removed. Under a nitrogen atmosphere, dichloromethane (69 mL) was added to the residue at 0°C, followed by trimethylsilyl cyanide (2.36 g) and stirring at 25°C for 96 hours. Tetra-n-butylammonium fluoride (1 mol / L tetrahydrofuran solution, 1 mL) was added to the reaction solution, which was then stirred for another 30 minutes, after which water was added and the mixture was extracted with chloroform. The organic layer was dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (elution solvent: hexane:ethyl acetate) to obtain compound A5 (2.74 g). 1H-NMR(400 MHz, CDCl3):1.64(3H,s), 1.65 (3H,s), 3.49-3.55 (1H,m), 3.73 (1H,dd, J = 10.9, 4.2 Hz), 3.79(1H,s), 3.80 (3H,s), 4.05 (1H,dd, J = 7.9, 3.6 Hz), 6.96-7.00 (2H,m),7.11(2H,d, J = 8.0 Hz), 7.21-7.40 (6H,m).

[0342] f) Preparation of Nα-[(1R)-2-hydroxy-1-phenylethyl]-β,β,1-trimethyl-L-tryptophanamide (Compound A6) To a suspension of compound A5 (2.74 g), dimethyl sulfoxide (6.16 g) and potassium carbonate (10.9 g) in methanol (50 mL) and water (2.1 mL), 30% hydrogen peroxide solution (8.94 mL) was added at 0° C., and the mixture was stirred at 45° C. for 1.5 hours. After the reaction was completed, a saturated aqueous solution of sodium thiosulfate was added, and the mixture was extracted with ethyl acetate. The organic layer was washed with water, dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (elution solvent: chloroform:methanol) to obtain compound A6 (2.32 g). 1 H-NMR(400 MHz, CDCl3):1.49(3H,s),1.51 (3H, s), 2.06-2.14 (1H, br), 2.37 (1H,dd, J = 6.0, 6.0 Hz),3.44-3.50 (1H, m),3.50-3.54 (1H, m), 3.56-3.63(m, 2H), 3.75 (3H, s), 5.52 (1H,brs), 6.14 (1H, brs), 6.71-6.73 (2H, m), 6.81-6.85(2H, m), 6.97-7.00 (2H, m),7.10-7.18 (2H, m), 7.24-7.28 (2H, m).

[0343] g) Preparation of β,β,1-trimethyl-L-tryptophanamide (Compound A7) Palladium hydroxide / carbon (2.8 g) was added to a solution of compound A6 (2.32 g) in methanol (65 mL), and the mixture was stirred at room temperature under a hydrogen atmosphere for 3 hours. After filtration through Celite, the solvent was distilled off, and the residue was purified by silica gel column chromatography (elution solvent: chloroform:methanol) to obtain compound A7 (1.27 g). 1 H-NMR(400 MHz, DMSO-d6):1.24(2H, brs), 1.28 (3H, s), 1.42(3H, s), 3.68 (1H, s), 3.71 (3H, s), 6.93-7.00(2H, m), 7.06 (1H, s), 7.11 (1H, t, J = 7.7 Hz), 7.29 (1H, brs), 7.36 (1H, d, J= 8.3 Hz), 7.88 (1H, d, J = 8.2Hz).

[0344] h) Preparation of Nα-(tert-butoxycarbonyl)-β,β,1-trimethyl-L-tryptophanamide (Compound A8) A mixture of compound A7 (1.27 g), sodium hydrogen carbonate (522 mg), di-tert-butyl dicarbonate (1.35 g), tetrahydrofuran (13 mL), chloroform (13 mL) and water (6.5 mL) was stirred at 25° C. for 16 hours. After the reaction was completed, water was added and the mixture was extracted with chloroform. The organic layer was dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (elution solvent: chloroform:methanol) to obtain compound A8 (1.80 g). 1H-NMR(400 MHz, CDCl3):1.33(3H, s), 1.47 (9H, s),1.50 (3H, s), 3.73 (3H, d, J = 1.3 Hz), 4.51 (1H, brs),4.86 (1H, brs), 5.02(1H, brd, J = 8.2 Hz), 5.59 (1H, brd, J = 6.4 Hz), 6.83(1H, d, J = 1.8 Hz),7.15 (1H, t, J = 7.3 Hz), 7.21-7.25 (1H, m), 7.30 (1H, d, J= 8.2 Hz), 8.05(1H, brd, J = 7.3 Hz). LC-MS: 346 (M+H) + (1.211 min, measurement condition A).

[0345] i) Preparation of N,N,Nα-tris(tert-butoxycarbonyl)-β,β,1-trimethyl-L-tryptophanamide (Compound A9) A mixture of compound A8 (1.79 g), di-tert-butyl dicarbonate (2.8 g), N,N-diisopropylethylamine (2.68 g), 4-dimethylaminopyridine (0.19 g) and chloroform (20 mL) was stirred at 25° C. for 2.5 hours. After the reaction was completed, water was added and the mixture was extracted with chloroform. The organic layer was washed with saturated saline, dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (elution solvent: hexane:ethyl acetate) to obtain compound A9 (1.99 g). 1 H-NMR(400 MHz, CDCl3):1.08-1.58(33H, m), 3.70(3H, s), 4.67-4.90 (0.2H, m), 5.25-5.45 (0.8H, m), 6.00-6.03 (1H,m), 6.81-6.87(1H, m), 7.04-7.09 (1H, m), 7.13-7.18 (1H. m), 7,21-7,27 (1H, m),7.91-7.94(1H, m). LC-MS: 546 (M+H) + (1.630 min, measurement condition A)

[0346] j) Preparation of methyl N-(tert-butoxycarbonyl)-β,β,1-trimethyl-L-tryptophanate (Compound A10) Under a nitrogen atmosphere, lithium methoxide (176 mg) was added to a methanol solution (21 mL) of compound A9 (2.29 g) at 0° C., and the mixture was stirred at 25° C. for 2 hours. After the reaction was completed, a saturated aqueous solution of ammonium chloride was added, and the mixture was extracted with ethyl acetate. The organic layer was washed with saturated saline, dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (elution solvent: hexane:ethyl acetate) to obtain compound A10 (927 mg). 1 H-NMR(400 MHz, CDCl3):1.17-1.59 (15H, m), 3.45 and 3.58 (3H, 2brs), 3.71(3H, s), 4.56-4.73 (1.2H, m),5.06 (0.8H, brd, J = 7.3 Hz), 6.81-6.82 (1H, m),7.05-7.10 (1H, m), 7.16-7.21(1H, m), 7.24-7.29 (1H, m), 7.73-7.80 (1H, m). LC-MS: 361 (M+H) + (1.379 min, measurement condition A).

[0347] k) Preparation of methyl N-(tert-butoxycarbonyl)-N,β,β,1-tetramethyl-L-tryptophanate (compound A11) Under a nitrogen atmosphere, 60% sodium hydride (168 mg) was added to a solution (13 mL) of compound A10 (927 mg) in N,N-dimethylformamide at 0° C., and then the mixture was stirred at 25° C. for 15 minutes. After the reaction suspension was cooled to 0° C., methyl iodide (1.1 g) was added, and the mixture was stirred at 25° C. for 1 hour. After the reaction was completed, water was added and the mixture was extracted with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (elution solvent: hexane:ethyl acetate) to obtain compound A11 (915 mg). 1 H-NMR(400 MHz, CDCl3):1.42(9H, s), 1.52 and 1.64 (6H, 2s), 2.80 and 2.86(3H, 2s), 3.46 (3H, s), 3.71 (3H,s), 5.27 and 5.52 (1H, 2s), 6.85 (1H, s),7.07-7.27(3H, m), 7.78 and 7.92 (1H,2d, J = 7.88 Hz). LC-MS: 397 (M+Na) + (1.406 min, measurement condition B)

[0348] l) Preparation of N-(tert-butoxycarbonyl)-N,β,β,1-tetramethyl-L-tryptophan (Compound A12) To a solution of compound A11 (639 mg) in water (11 mL)-methanol (44 mL), 1 mol / L lithium hydroxide (13.5 mL) was added and stirred at 60° C. for 24 hours. After the reaction was completed, a 1 mol / L aqueous oxalic acid solution was added to adjust the reaction solution to pH 4, water was added, and the solution was extracted with chloroform. The organic layer was washed with saturated saline, dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (elution solvent: chloroform:methanol) to obtain compound A12 (610 mg). 1 H-NMR(400 MHz, CDCl3):1.43(9H, s), 1.53 (3H, s), 1.63 (3H, s), 2.76 and2.89 (3H, 2s), 3.71 (3H, s), 5.36and 5.44 (1H, 2s), 6.85 and 6.87 (1H, 2s),7.02-7.11 (1H, m), 7.18 (1H, t, J =7.3 Hz), 7.24-7.27 (1H, m), 7.81 and 7.96 (1H,2d, J = 7.9 Hz). LC-MS: 361 (M+H) + , 359 (MH) - (1.300 min, measurement condition A).

[0349] m) Preparation of N-(tert-butoxycarbonyl)-N,β,β,1-tetramethyl-L-tryptophyl-N-[(3S,4E)-6-ethoxy-2,5-dimethyl-6-oxohex-4-en-3-yl]-N,3-dimethyl-L-valinamide (Compound A13) A mixture of compound A12 (500 mg), ethyl (2E,4S)-2,5-dimethyl-4-[methyl(3-methyl-L-valyl)amino]hex-2-enoate (520 mg), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (399 mg), 1-hydroxy-1H-benzotriazole monohydrate (425 mg) and N,N-dimethylformamide (5 mL) was stirred at 25°C for 16 hours. After the reaction was completed, water was added and the mixture was extracted with chloroform. The organic layer was washed with saturated saline, dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (elution solvent: hexane:ethyl acetate) to obtain compound A13 (759 mg). LC-MS: 655 (M+H) + (1.714 min, measurement condition A)

[0350] n) Preparation of N-(tert-butoxycarbonyl)-N,β,β,1-tetramethyl-L-tryptophyll-N-[(3S,4E)-5-carboxy-2-methylhex-4-en-3-yl]-N,3-dimethyl-L-valinamide (Compound A14) 1 mol / L lithium hydroxide (1.65 mL) was added to a solution of compound A13 (127 mg) in water (1.55 mL)-methanol (4.65 mL), and the mixture was stirred at 25° C. for 24 hours. After the reaction was completed, 1 mol / L aqueous oxalic acid was added to adjust the reaction solution to pH 4, and then water was added and the mixture was extracted with chloroform. The organic layer was washed with saturated saline, dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (elution solvent: chloroform:methanol) to obtain compound A14 (93 mg). LC-MS: 627 (M+H) + (1.508 min, measurement condition A)

[0351] o) Preparation of N-(tert-butoxycarbonyl)-N,β,β,1-tetramethyl-L-tryptophyl-N-{(3S,4E)-6-[(2,5-dioxopyrrolidin-1-yl)oxy]-2,5-dimethyl-6-oxohex-4-en-3-yl}-N,3-dimethyl-L-valinamide (Compound A15) A mixture of compound A14 (185 mg), N-hydroxysuccinimide (97 mg), bromotripyrrolidinophosphonium hexafluorophosphate (391 mg), 4-dimethylaminopyridine (102 mg), N,N-diisopropylethylamine (108 mg) and N,N-dimethylformamide (2.8 mL) was stirred at 25° C. for 4 hours. After the reaction was completed, water was added and the mixture was extracted with ethyl acetate. The organic layer was washed with water, dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (elution solvent: chloroform:methanol) to obtain compound A15 (166 mg). 1 H-NMR(400 MHz, CDCl3):8.27 and 7.96 (1H, 2d, J = 7.9 Hz), 7.16-7.04 (4H,m), 6.88 (1H, d, J = 9.1 Hz),6.17 and 6.09 (1H, 2d, J = 8.5 Hz), 5.96 and 5.66(1H, 2s), 5.07 (1H, t, J = 9.3Hz), 4.45 and 3.87 (1H, 2d, J = 8.6 Hz), 3.74and 3.73 (3H, 2s), 2.99 (3H, s),2.95 (3H, s), 2.83 (4H, brs), 1.97 (3H, s),1.92-1.86 (1H, m), 1.57-1.42 (14H,m), 0.89 (3H, d, J = 6.1 Hz), 0.83-0.80 (3H,m), 0.48 and 0.41 (9H, 2s). LC-MS: 724 (M+H) + (1.573 min, measurement condition A)

[0352] p) Preparation of (6S,9S,12S,13E,17R)-17-(tert-butoxycarbonyl)-9-tert-butyl-2,2,5,11,14-pentamethyl-6-[2-(1-methyl-1H-indol-3-yl)propan-2-yl]-4,7,10,15-tetraoxo-12-(propan-2-yl)-3-oxa-5,8,11,16-tetraazaicos-13-ene-20-carboxylic acid (compound A16) A mixture of compound 15 (30 mg), D-glutamic acid α-tert-butyl ester hydrochloride (10.7 mg), N,N-diisopropylethylamine (49.7 mg) and N,N-dimethylformamide (1.0 mL) was stirred at 25° C. for 3 hours. After the reaction was completed, water was added and the mixture was extracted with chloroform. The organic layer was washed with water, dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (elution solvent: chloroform:methanol) to obtain compound A16 (14.2 mg). LC-MS 834 (M+Na) + (1.574 min, measurement condition D)

[0353] q) Preparation of tert-butyl (6S,9S,12S,13E,17R)-9-tert-butyl-17-(3-{[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]amino}-3-oxopropyl)-2,2,5,11,14-pentamethyl-6-[2-(1-methyl-1H-indol-3-yl)propan-2-yl]-4,7,10,15-tetraoxo-12-(propan-2-yl)-3-oxa-5,8,11,16-tetraazaoctadec-13-ene-18-carboxylate (Reference Example 1) A mixture of compound A16 (14 mg), N-(2-aminoethyl)maleimide hydrochloride (3.0 mg), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (6.6 mg), 1-hydroxy-1H-benzotriazole monohydrate (5.2 mg), N,N-diisopropylethylamine (4.4 mg) and N,N-dimethylformamide (0.5 mL) was stirred at 25°C for 2 hours. After the reaction was completed, water was added and the mixture was extracted with ethyl acetate. The organic layer was washed with water, dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (elution solvent: chloroform:methanol) to obtain compound A15 (166 mg). LC-MS: 934 (M+H) + (1.597 min, measurement condition D)

[0354] Reference example 2 N-(tert-butoxycarbonyl)-N,β,β-trimethyl-L-phenylalanyl-N-{(3S,4E)-6-[(2,5-dioxopyrrolidin-1-yl)oxy]-2,5-dimethyl-6-oxohex-4-en-3-yl}-N,3-dimethyl-L-valinamide [ka]

[0355] a) Preparation of 3-methyl-3-phenylbutanoic acid (compound J1) To a solution of 3-methyl-2-butenoic acid (15 g) in benzene (100 mL), aluminum chloride (24.1 g) was added at 10° C., and the mixture was stirred for 30 minutes and then at 40° C. for 1 hour. After cooling to 0° C., ice water was added, the mixture was extracted with tert-butyl methyl ether, and concentrated to a certain extent, and the organic layer was extracted with a saturated aqueous solution of sodium bicarbonate. The aqueous layer was adjusted to pH 2 with concentrated hydrochloric acid, and extracted with tert-butyl methyl ether. The organic layer was dried over anhydrous magnesium sulfate, and the solvent was distilled off under reduced pressure to obtain compound J1 (26.3 g). 1H-NMR(400 MHz, CDCl3):1.46(6H, s), 2.65 (2H, s),7.20 (1H, t, J = 7.2 Hz), 7.31 (1H, t, J = 7.2 Hz), 7.37(2H, d, J = 7.2 Hz).

[0356] b) Preparation of (4S)-3-(3-methyl-3-phenylbutanoyl)-4-(propan-2-yl)-1,3-oxazolidin-2-one (compound J2) To a THF solution (900 mL) of compound J1 (17.2 g), triethylamine (23.7 mL) and pivaloyl chloride (15.3 mL) were added at -78°C. The mixture was heated to 0°C and stirred for 1 hour. Separately, n-butyllithium (89.8 mL of 1.64 mol / L hexane solution) was added to a THF solution (760 mL) of (S)-isopropyloxazolidinone (19.5 g) at -78°C and stirred for 30 minutes to prepare a lithium salt. The reaction solution was cooled to -78°C, the lithium salt was added dropwise, and the mixture was stirred for 1 hour, then heated to 0°C and stirred for another 30 minutes, after which water was added and the mixture was extracted with tert-butyl methyl ether. The organic layer was washed with saturated saline and dried over anhydrous magnesium sulfate. The solvent was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography (elution solvent: hexane: tert-butyl methyl ether) to obtain compound J2 (27.0 g). 1 H-NMR(400 MHz, CDCl3):0.723(3H, d, J = 6.8 Hz),0.80 (3H, d, J = 6.8 Hz), 1.49 (s, 6H), 2.13-2.18 (m, 1H),3.36 (s, 3H),3.99-4.09 (m, 2H), 4.20-4.23 (m, 1H), 7.16-7.20 (m, 1H), 7.28-7.32(m, 2H),7.38-7.40 (m, 2H).

[0357] c) Preparation of (4S)-3-[(2S)-2-azido-3-methyl-3-phenylbutanoyl]-4-(propan-2-yl)-1,3-oxazolidin-2-one (compound J3) A suspension of compound J2 (27.0 g) in THF (560 mL) was cooled to -78°C, potassium hexamethyldisilazide (1.06 mol / L tetrahydrofuran solution, 99.5 mL) was added, and the mixture was stirred for 1.5 hours. A solution of 2,4,6-triisopropylbenzenesulfonyl azide (40 g) in THF (330 mL) at -78°C was added, and after 10 minutes, acetic acid (24.5 mL) was added, and the mixture was heated to 40°C and stirred for 1 hour. Saturated saline was added, and the mixture was extracted with tert-butyl methyl ether. The organic layer was washed with a saturated aqueous sodium bicarbonate solution and saturated saline, and then the organic layer was dried over anhydrous magnesium sulfate. The solvent was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography (elution solvent: hexane:chloroform) to obtain compound J3 (16.4 g). 1 H-NMR(400 MHz, CDCl3):0.80(3H, d, J = 6.8 Hz),0.84 (3H, d, J = 7.2 Hz), 1.54 (3H, s), 1.56 (3H, s),2.28-2.33 (1H, m),3.54-3.59 (1H, m), 3.87-3.90 (1H, m), 3.95-3.98 (1H, m), 5.66(1H, s),7.23-7.420 (5H, m).

[0358] d) Preparation of tert-butyl {(2S)-3-methyl-1-oxo-1-[(4S)-2-oxo-4-(propan-2-yl)-1,3-oxazolidin-3-yl]-3-phenylbutan-2-yl}carbamate (compound J4) To a solution (1200 mL) of compound J3 (16.4 g) in ethyl acetate, di-tert-butyl dicarbonate (24.0 g) and 10% Pd-C (11.6 g, 50% wet) were added, and the mixture was stirred under a hydrogen atmosphere for 2 hours. The mixture was filtered through Celite and washed with ethyl acetate. The solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography (elution solvent: hexane: tert-butyl methyl ether) to obtain compound J4 (16.1 g). 1H-NMR(400 MHz, CDCl3):0.77(3H, d, J = 6.8 Hz),0.82 (3H, d, J = 6.8 Hz), 1.42 (3H, s), 1.43 (9H, s), 1.48(3H, s), 2.20-2.29(1H, m), 3.45 (1H, t, J = 8.8 Hz), 3.80-3.83 (1H, m),3.89-3.92 (1H, dd, J =2.0 Hz, J = 8.4 Hz), 5.16 (1H, brs), 6.13 (1H, d, J = 9.6Hz), 7.21-7.26 (1H, m), 7.29-7.33 (2H, m). 7.42 (2H, d, J = 7.2 Hz).

[0359] e) Preparation of N-(tert-butoxycarbonyl)-β,β-dimethyl-L-phenylalanine (Compound J5) To a solution of compound J4 (16.1 g) in THF (468 mL) and water (117 mL), 30% hydrogen peroxide solution (32.5 mL) and lithium hydroxide solution (1 mol / L, 119 mL) were added at 0° C., the temperature was raised to 25° C., and the mixture was stirred for 3 hours. An aqueous sodium hydrogen sulfate solution (1.5 mol / L, 470 mL) was added at 0° C., the temperature was raised to 25° C., and the mixture was stirred for 1 hour. The pH was adjusted to 3 with an aqueous citric acid solution (1 mol / L), and the mixture was extracted with tert-butyl methyl ether. The organic layer was washed with saturated saline, and the organic layer was dried over anhydrous magnesium sulfate. The solvent was distilled off under reduced pressure to obtain compound J5 (14.2 g). 1 H-NMR(400 MHz, CDCl3):1.38(9H, s), 1.44 (3H, s),1.46 (3H, s), 4.56 (1H, brd, J = 11.6 Hz), 4.94 (1H, brd,J = 14.4 Hz),7.21-7.38 (5H, m).

[0360] f) Preparation of methyl N-(tert-butoxycarbonyl)-β,β-dimethyl-L-phenylalanine ester (Compound J6) Sodium carbonate (8.44 g) and methyl iodide (9.91 mL) were added to a solution (84 mL) of compound J5 (14.2 g) in N,N-dimethylformamide, and the mixture was stirred at 25° C. for 15 hours. After cooling to 0° C., cold water was added and the mixture was extracted with tert-butyl methyl ether. The organic layer obtained was washed with saturated saline and then dried over anhydrous magnesium sulfate. The solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography (elution solvent: hexane: tert-butyl methyl ether) to obtain compound J6 (11.1 g). 1 H-NMR(400 MHz, CDCl3):1.36(9H, s), 1.37 (3H, s),1.41 (3H, s), 3.48 (3H, brs), 4.49 (1H, brd, J = 9.8 Hz),4.98 (1H, brd, J =9.1 Hz), 7.18-7.22 (1H, m), 7.27-7.33 (4H, m).

[0361] g) Preparation of methyl N-(tert-butoxycarbonyl)-N,β,β-trimethyl-L-phenylalanine ester (Compound J7) Compound J7 (245 mg) was obtained from compound J6 (307 mg) in the same manner as in Reference Example 1-k). LC-MS: 344 (M+Na) + (1.589 min, measurement condition C)

[0362] h) Preparation of N-(tert-butoxycarbonyl)-N,β,β-trimethyl-L-phenylalanine (Compound J8) Compound J8 (195 mg) was obtained from compound J7 (235 mg) in the same manner as in Reference Example 1-l). LC-MS: 330 (M+Na) + (1.420 min, measurement condition C)

[0363] i) Preparation of N-(tert-butoxycarbonyl)-N,β,β-trimethyl-L-phenylalanyl-N-[(3S,4E)-6-ethoxy-2,5-dimethyl-6-oxohex-4-en-3-yl]-N,3-dimethyl-L-valinamide (Compound J9) Compound J9 (307 mg) was obtained from compound J8 (195 mg) in the same manner as in Reference Example 1-m). LC-MS: 624 (M+Na) + (1.797 min, measurement condition C)

[0364] j) Preparation of N-(tert-butoxycarbonyl)-N,β,β-trimethyl-L-phenylalanyl-N-[(3S,4E)-5-carboxy-2-methylhex-4-en-3-yl]-N,3-dimethyl-L-valinamide (Compound J10) Compound J10 (286 mg) was obtained from compound J9 (307 mg) in the same manner as in Reference Example 1-n). LC-MS: 596 (M+Na) + , 572 (MH) - (1.596 min, measurement condition C)

[0365] k) Preparation of N-(tert-butoxycarbonyl)-N,β,β-trimethyl-L-phenylalanyl-N-{(3S,4E)-6-[(2,5-dioxopyrrolidin-1-yl)oxy]-2,5-dimethyl-6-oxohex-4-en-3-yl}-N,3-dimethyl-L-valinamide (Reference Example 2) Reference Example 2 (227 mg) was obtained from Compound J10 (286 mg) in the same manner as in Reference Example 1-o). LC-MS: 693 (M+Na) + (1.658 min, measurement condition C)

[0366] Reference example 3 Di-tert-butyl(6S,9S,12S,13E,17R,22R)-9-tert-butyl-28-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2,2,5,11,14-pentamethyl-6-[2-(1-methyl-1H-indol-3-yl)propan-2-yl]-4,7,10,15,20,25-hexaoxo-12-(propan-2-yl)-3-oxa-5,8,11,16,21,26-hexaazaoctacos-13-ene-17,22-carboxylic acid diester [ka]

[0367] a) Preparation of tert-butyl (6S,9S,12S,13E,17R)-9-tert-butyl-17-{3-[(2,5-dioxopyrrolidin-1-yl)oxy]-3-oxopropyl}-2,2,5,11,14-pentamethyl-6-[2-(1-methyl-1H-indol-3-yl)propan-2-yl]-4,7,10,15-tetraoxo-12-(propan-2-yl)-3-oxa-5,8,11,16-tetraazaoctadec-13-ene-18-carboxylate (compound C1) Compound C1 (51.8 mg) was obtained from compound A16 (90.2 mg) by the same method as in Reference Example 1-o). LC-MS: 931 (M+Na) + (1.662 min, measurement condition D)

[0368] b) Preparation of (6S,9S,12S,13E,17R,22R)-17,22-bis(tert-butoxycarbonyl)-9-tert-butyl-2,2,5,11,14-pentamethyl-6-[2-(1-methyl-1H-indol-3-yl)propan-2-yl]-4,7,10,15,20-pentaoxo-12-(propan-2-yl)-3-oxa-5,8,11,16,21-pentaazapentacos-13-ene-25-carboxylic acid (compound C2) Compound C2 (40 mg) was obtained from compound C1 (51.8 mg) by the same method as in Reference Example 1-p). LC-MS: 1019 (M+Na) + (1.422 min, measurement condition D)

[0369] c) Preparation of di-tert-butyl (6S,9S,12S,13E,17R,22R)-9-tert-butyl-28-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2,2,5,11,14-pentamethyl-6-[2-(1-methyl-1H-indol-3-yl)propan-2-yl]-4,7,10,15,20,25-hexaoxo-12-(propan-2-yl)-3-oxa-5,8,11,16,21,26-hexaazaoctacos-13-ene-17,22-carboxylic acid diester (Reference Example 3) Reference Example 3 (14 mg) was obtained from Compound C2 (40 mg) by the same method as in Reference Example 1-q). LC-MS: 1141 (M+Na) + (1.618 min, measurement condition D)

[0370] Reference example 4 N-{(2E,4S)-2,5-dimethyl-4-[methyl(N,β,β,1-tetramethyl-L-tryptophyll-3-methyl-L-valyl)amino]hex-2-enoyl}-D-γ-glutamyl-L-lysine [ka]

[0371] a) Preparation of (6S,9S,12S,13E,17R)-9-tert-butyl-17-(ethoxycarbonyl)-2,2,5,11,14-pentamethyl-6-[2-(1-methyl-1H-indol-3-yl)propan-2-yl]-4,7,10,15-tetraoxo-12-(propan-2-yl)-3-oxa-5,8,11,16-tetraazaicos-13-ene-20-carboxylic acid (Compound D1) A mixture of compound A15 (160 mg), α-ethyl D-glutamic acid ester trifluoroacetate (122 mg), N,N-diisopropylethylamine (100 mg) and N,N-dimethylformamide (2.2 mL) was stirred at 25°C for 6 hours. After the reaction was completed, the pH was adjusted to 4 with 1 mol / L aqueous oxalic acid solution and extracted with chloroform. The organic layer was washed with water and saturated saline, dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (elution solvent: chloroform:methanol) to obtain compound D1 (155 mg). 1 H-NMR(400 MHz, CDCl3):8.26 and 7.97 (1H, 2d, J = 7.9 Hz), 7.32-7.05 (4H,m), 6.71 (1H, t, J = 6.7 Hz),6.45 (1H, d, J = 8.6 Hz), 6.31-6.26 (1H, m), 5.95and 5.63 (1H, 2s), 4.94-4.82(1H, m), 4.64-4.59 (1H, m), 4.51 and 4.41 (1H, 2d,J = 9.1 Hz), 4.21 (2H, q, J =7.3 Hz), 3.75 and 3.74 (3H, 2s), 3.00 (3H, s),2.97 and 2.95 (3H, 2s), 2.52-2.38(2H, m), 2.29-2.20 (1H, m), 2.10-2.00 (1H,m), 1.98-1.90 (1H, m), 1.90 (3H, s),1.57-1.45 (14H, m), 1.28 (3H, t, J = 7.3Hz), 0.88 (3H, d, J = 6.1 Hz), 0.82(3H, d, J = 6.7 Hz), 0.53 and 0.46 (9H,2s). LC-MS 784 (M+H) + , 782 (MH) - (1.472 min, measurement condition A)

[0372] b) Preparation of 17-ethyl 22-methyl(6S,9S,12S,13E,17R,22S)-9-tert-butyl-2,2,5,11,14,30,30-heptamethyl-6-[2-(1-methyl-1H-indol-3-yl)propan-2-yl]-4,7,10,15,20,28-hexaoxo-12-(propan-2-yl)-3,29-dioxa-5,8,11,16,21,27-hexaazahentriacont-13-ene-17,22-dicarboxylate (compound D2) Compound D2 (13 mg) was obtained from compound D1 (20 mg) in the same manner as in Reference Example 1-m). LC-MS: 1026 (M+H) + (1.597 min, measurement condition D)

[0373] c) Preparation of 14-ethyl 19-methyl(3S,6S,9S,10E,14R,19S)-23-amino-6-tert-butyl-8,11-dimethyl-3-[2-(1-methyl-1H-indol-3-yl)propan-2-yl]-4,7,12,17-tetraoxo-9-(propan-2-yl)-2,5,8,13,18-pentaazatricos-10-ene-14,19-dicarboxylate (compound D3) Trifluoroacetic acid (0.2 mL) was added to a chloroform solution (1.0 mL) of compound D2 (13 mg) and the mixture was stirred for 1 hour at 25° C. After completion of the reaction, the reaction solution was purified by silica gel column chromatography (elution solvent: methanol:chloroform) to obtain compound D3 (10 mg). LC-MS: 826 (M+H) + , 824 (MH) - (0.978 min, measurement condition D)

[0374] d) Preparation of N-{(2E,4S)-2,5-dimethyl-4-[methyl(N,β,β,1-tetramethyl-L-tryptophyll-3-methyl-L-valyl)amino]hex-2-enoyl}-D-γ-glutamyl-L-lysine (Reference Example 4) Reference Example 4 (7.2 mg) was obtained from Compound D3 (10 mg) by preparing the compound in the same manner as in Reference Example 1-n) and purifying the compound by reverse phase column chromatography (elution solvent: 0.1% TFA acetonitrile:water). LC-MS: 784 (M+H) + , 782 (MH) - (0.889 min, measurement condition D)

[0375] Reference example 5 tert-Butyl N5-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)-D-glutamate [ka]

[0376] a) Preparation of tert-butyl N2-(tert-butoxycarbonyl)-N5-(2-(2,5-dioxy-2,5-dihydro-1H-pyrrol-1-yl)ethyl)-D-glutamate A mixture of BOC-D-glutamic acid α-tert-butyl ester (2.061 g), 1-(2-amino-ethyl)-pyrrole-2,5-dione hydrochloride (1.20 g), 2-(1H-benzo[d][1,2,3]triazol-1-yl)-1,1,3,3-tetramethylisouronium hexafluorophosphate (V) (3.87 g), N,N-diisopropylethylamine (3.47 mL) and N,N-dimethylformamide (10 mL) was stirred at room temperature for 1 hour. After the reaction was completed, ethyl acetate was added, and the organic layer was washed with saturated sodium bicarbonate solution and saturated saline, then dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The residue was purified by silica gel chromatography (eluent: hexane:ethyl acetate) to give tert-butyl N2-(tert-butoxycarbonyl)-N5-(2-(2,5-dioxy-2,5-dihydro-1H-pyrrol-1-yl)ethyl)-D-glutamate (2.8 g). LC-MS: 426 (M+H) + (1.030 min, measurement condition F)

[0377] b) Preparation of tert-butyl N5-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)-D-glutamate (Reference Example 5) A mixture of tert-butyl N2-(tert-butoxycarbonyl)-N5-(2-(2,5-dioxy-2,5-dihydro-1H-pyrrol-1-yl)ethyl)-D-glutamate (51.8 mg) and TFA (1 mL) was stirred at room temperature for 1 hour and 20 minutes. The reaction solution was cooled on ice and concentrated under reduced pressure to obtain Reference Example 5. The compound was used in the next reaction without purification. LC-MS: 326 (M+H) + (0.496min, measurement condition F)

[0378] Reference example 6 N5-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)-D-glutamine [ka]

[0379] A mixture of tert-butyl N2-(tert-butoxycarbonyl)-N5-(2-(2,5-dioxy-2,5-dihydro-1H-pyrrol-1-yl)ethyl)-D-glutamate (64.8 mg) and TFA (1 mL) was stirred at room temperature for 17 hours. After completion of the reaction, the mixture was concentrated under reduced pressure to obtain Reference Example 6. The compound was used in the next reaction without purification. LC-MS: 270 (M+H) + (0.254min, measurement condition F)

[0380] Reference example 7 N-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]-D-glutaminyl-D-glutamic acid [ka]

[0381] a) Preparation of di-tert-butyl ((R)-2-((tert-butoxycarbonyl)amino)-5-methoxy-5-oxopentanoyl)-D-glutamate A mixture of N-α-(T-butoxycarbonyl)-D-glutamic acid γ-methyl ester (261 mg), D-glutamic acid di-tert-butyl ester hydrochloride (295 mg), 2-(1H-benzo[d][1,2,3]triazol-1-yl)-1,1,3,3-tetramethylisouronium hexafluorophosphate (V) (456 mg), N,N-diisopropylethylamine (0.446 mL) and N,N-dimethylformamide (4 mL) was stirred at room temperature for 2 hours. After the reaction was completed, water was added and the mixture was extracted with ethyl acetate. The organic layer was washed with water and a saturated aqueous solution of sodium bicarbonate, dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The residue was purified by silica gel chromatography (eluent: hexane:ethyl acetate) to give (di-tert-butyl ((R)-2-((tert-butoxycarbonyl)amino)-5-methoxy-5-oxopentanoyl)-D-glutamate (502 mg). 1 H-NMR (400 MHz, CDCl3):1.42(18H, s), 1.44 (9H, s), 1.84-1.94 (2H, m), 2.12 (2H, dtt, J = 22.5, 8.4, 3.0Hz), 2.26 (2H, dtd, J = 25.2, 10.0, 4.5 Hz), 2.43 (2H, tdd, J = 24.8, 14.2, 7.5Hz), 3.67 (3H, s), 4.14 (1H, t, J = 6.1 Hz), 4.43 (1H, td, J = 7.9, 4.9 Hz),5.23 (1H, d, J = 7.3 Hz), 6.81 (1H, d, J = 7.3 Hz).

[0382] b) Preparation of (R)-4-((tert-butoxycarbonyl)amino)-5-(((R)-1,5-di-tert-butoxy-1,5-dioxopentan-2-yl)amino)-5-oxopentanoic acid A mixture of di-tert-butyl ((R)-2-((tert-butoxycarbonyl)amino)-5-methoxy-5-oxopentanoyl)-D-glutamate (502 mg), 1 mol / L aqueous lithium hydroxide solution (0.999 mL) and methanol (5 mL) was stirred at room temperature for 16 hours. After the reaction was completed, 1 mol / L aqueous citric acid solution was added to make an acidic (pH 4) solution, and the methanol was distilled off under reduced pressure, followed by extraction with chloroform. The organic layer was dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The residue was purified by silica gel chromatography (elution solvent: chloroform:methanol) to give (R)-4-((tert-butoxycarbonyl)amino)-5-(((R)-1,5-di-tert-butoxy-1,5-dioxopentan-2-yl)amino)-5-oxopentanoic acid (393 mg). LC-MS: 489 (M+H) + (1.417 min, measurement condition G)

[0383] c) Preparation of di-tert-butyl N2-(tert-butoxycarbonyl)-N5-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)-D-glutaminyl-D-glutamate A mixture of (R)-4-((tert-butoxycarbonyl)amino)-5-(((R)-1,5-di-tert-butoxy-1,5-dioxopentan-2-yl)amino)-5-oxopentanoic acid (205 mg), 1-(2-amino-ethyl)-pyrrole-2,5-dione hydrochloride (89 mg), 2-(1H-benzo[d][1,2,3]triazol-1-yl)-1,1,3,3-tetramethylisouronium hexafluorophosphate (V) (191 mg), N,N-diisopropylethylamine (0.187 mL) and N,N-dimethylformamide (3 mL) was stirred at room temperature for 16 hours. After completion of the reaction, water was added and the mixture was extracted with chloroform. The organic layer was dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The residue was purified by silica gel chromatography (eluent: chloroform:methanol) to give di-tert-butyl N2-(tert-butoxycarbonyl)-N5-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)-D-glutaminyl-D-glutamate (13 mg). LC-MS: 611 (M+H) + (1.638min, measurement condition G)

[0384] d) Preparation of N-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]-D-glutaminyl-D-glutamic acid (Reference Example 7) Di-tert-butyl N2-(tert-butoxycarbonyl)-N5-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)-D-glutaminyl-D-glutamate (13 mg) was reacted and treated in the same manner as in Example M1 to synthesize Reference Example 7. The synthesized Reference Example 7 was used in the next reaction as it was without purification.

[0385] Reference examples 8~18 According to the method described in the literature (Bioorg Med Chem Lett. 2004 Nov 1;14(21):5317-22, J Med Chem. 2004 Sep 9;47(19):4774-86, WO 2003 / 082268 and WO 2016 / 123582), the compounds shown in Table 1 below were obtained. [Table 1-1] [Table 1-2]

[0386] Reference example 19 N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino)-3-(m-tolyl)butanamide)hexa-2-enoic acid [ka]

[0387] A mixture of (S,E)-4-((S)-2-((S)-3-(3-bromophenyl)-3-methyl-2-(methylamino)butanamido)-N,3,3-trimethylbutanamido)-2,5-dimethylhex-2-enoic acid (62.5 mg), tetrakis(triphenylphosphine)palladium(0) (13.07 mg), dimethylzinc (0.113 mL) and tetrahydrofuran (5 mL) was stirred at 60° C. for 2.5 hours. After completion of the reaction, the solvent was distilled off under reduced pressure. The residue was purified by silica gel chromatography (elution solvent: chloroform:methanol) to obtain Reference Example 19 (29.7 mg). LC-MS: 488 (M+H) + (0.68 min, measurement condition F)

[0388] Reference example 20 (S,E)-4-((S)-2-((S)-3-(3-cyanophenyl)-3-methyl-2-(methylamino)butanamido)-N,3,3-trimethylbutanamido)-2,5-dimethylhex-2-enoic acid [ka]

[0389] A mixture of (S,E)-4-((S)-2-((S)-3-(3-bromophenyl)-3-methyl-2-(methylamino)butanamido)-N,3,3-trimethylbutanamido)-2,5-dimethylhex-2-enoic acid (76.1 mg), tetrakis(triphenylphosphine)palladium(0) (15.92 mg), zinc (18.01 mg), zinc cyanide (32.3 mg) and N,N-dimethylformamide (1 mL) was stirred at 120° C. for 1 hour under microwave irradiation. After completion of the reaction, the solvent was distilled off under reduced pressure. The residue was roughly purified by silica gel chromatography (elution solvent: chloroform:methanol), and then the product of Reference Example 20 (53.5 mg) was obtained by reverse phase HPLC (mobile phase: 0.1% TFA water / 0.035% TFA acetonitrile solvent). LC-MS: 499 (M+H) + (0.99 min, measurement condition F)

[0390] Reference example 21 (S,E)-4-((S)-2-((S)-3-([1,1'-biphenyl]-3-yl)-3-methyl-2-(methylamino)butanamido)-N,3,3-trimethylbutanamido)-2,5-dimethylhex-2-enoic acid [ka]

[0391] A mixture of (S,E)-4-((S)-2-((S)-3-(3-bromophenyl)-3-methyl-2-(methylamino)butanamido)-N,3,3-trimethylbutanamido)-2,5-dimethylhex-2-enoic acid (64.3 mg), tetrakis(triphenylphosphine)palladium(0) (13.45 mg), phenylboronic acid (28.4 mg), sodium carbonate (24.67 mg) and tetrahydrofuran (5 mL) was stirred at 80° C. for 3.5 hours. The solvent was distilled off under reduced pressure. Reference Example 21 (10.7 mg) was obtained by reverse phase HPLC (mobile phase: 0.1% TFA water / 0.035% TFA acetonitrile solvent). LC-MS: 550 (M+H) + (0.88min, measurement condition F)

[0392] Reference example 22 4-(4-(((S)-1-(((S,E)-5-carboxy-2-methylhex-4-en-3-yl)(methyl)amino)-3,3-dimethyl-1-oxabutan-2-yl)amino)-2-methyl-3-(methylamino)-4-oxobutan-2-yl)benzoic acid [ka]

[0393] a) Preparation of 4-(4-(((S)-1-(((S,E)-6-ethoxy-2,5-dimethyl-6-oxohex-4-en-3-yl)(methyl)amino)-3,3-dimethyl-1-oxabutan-2-yl)amino)-2-methyl-3-(methylamino)-4-oxabutan-2-yl)benzoic acid A mixture of ethyl (9S,12S,E)-6-(2-(4-(tert-butoxycarbonyl)phenyl)propan-2-yl)-9-(tert-butyl)-12-isopropyl-2,2,5,11,14-pentamethyl-4,7,10-trioxo-3-oxa-5,8,11-triazapentadec-13-en-15-oate (400 mg), TFA (2 mL) and chloroform (8 mL) was stirred at room temperature for 4 hours. The solvent was distilled off under reduced pressure to give 4-(4-(((S)-1-(((S,E)-6-ethoxy-2,5-dimethyl-6-oxohex-4-en-3-yl)(methyl)amino)-3,3-dimethyl-1-oxabutan-2-yl)amino)-2-methyl-3-(methylamino)-4-oxabutan-2-yl)benzoic acid (376 mg). The compound obtained was used in the next reaction without purification. LC-MS: 546 (M+H) + (1.15 min, measurement condition F)

[0394] b) Preparation of 4-(4-(((S)-1-(((S,E)-5-carboxy-2-methylhex-4-en-3-yl)(methyl)amino)-3,3-dimethyl-1-oxabutan-2-yl)amino)-2-methyl-3-(methylamino)-4-oxobutan-2-yl)benzoic acid (Reference Example 22) A mixture of 4-(4-(((S)-1-(((S,E)-6-ethoxy-2,5-dimethyl-6-oxohex-4-en-3-yl)(methyl)amino)-3,3-dimethyl-1-oxabutan-2-yl)amino)-2-methyl-3-(methylamino)-4-oxabutan-2-yl)benzoic acid (55.2 mg), methanol (3 mL) and water (1 mL) was stirred under ice cooling, and lithium hydroxide (16.98 mg) was added. The mixture was stirred at room temperature for 2 days. The solvent was distilled off under reduced pressure. The residue was subjected to reverse phase HPLC (mobile phase: 0.1% TFA water / 0.035% TFA acetonitrile solvent) to obtain Reference Example 22 (21.1 mg). LC-MS: 518 (M+H) + (1.18min, measurement condition F)

[0395] Reference example 23 (4S,E)-4-((2S)-2-(3-(4-(tert-butoxycarbonyl)phenyl)-3-methyl-2-(methylamino)butanamide)-N,3,3-trimethylbutanamide)-2,5-dimethylhex-2-enoic acid [ka] The compound shown in Reference Example 23 was obtained from tert-butyl 4-(4-(((S)-1-(((S,E)-6-ethoxy-2,5-dimethyl-6-oxohex-4-en-3-yl)(methyl)amino)-3,3-dimethyl-1-oxobutan-2-yl)amino)-2-methyl-3-(methylamino)-4-oxobutan-2-yl)benzoate in the same manner as in step n) of Reference Example 1. LC-MS: 574 (M+H) + (1.29 min, measurement condition G)

[0396] Reference example 24 (S,E)-4-((S)-2-((S)-3-(4-(tert-butoxycarbonyl)phenyl)-3-methyl-2-(methoxyamino)butanamido)-N,3,3-trimethylbutanamido)-2,5-dimethylhex-2-enoic acid [ka]

[0397] a) Preparation of tert-butyl 4-((S)-4-(((S)-1-(((S,E)-6-ethoxy-2,5-dimethyl-6-oxohex-4-en-3-yl)(methyl)amino)-3,3-dimethyl-1-oxobutan-2-yl)amino)-2-methyl-3-(methylamino)-4-oxobutan-2-yl)benzoate To a mixture of 4-(4-(((S)-1-(((S,E)-6-ethoxy-2,5-dimethyl-6-oxohex-4-en-3-yl)(methyl)amino)-3,3-dimethyl-1-oxobutan-2-yl)amino)-2-methyl-3-(methylamino)-4-oxobutan-2-yl)benzoic acid (104.0 mg) and toluene (1 mL), N,N-dimethylfluoroamido-di-tert-butyl acetate (0.456 mL) was added and the mixture was heated to reflux for 14 hours, and then the solvent was distilled off under reduced pressure. The residue was purified by silica gel chromatography to obtain tert-butyl 4-((S)-4-(((S)-1-(((S,E)-6-ethoxy-2,5-dimethyl-6-oxohex-4-en-3-yl)(methyl)amino)-3,3-dimethyl-1-oxobutan-2-yl)amino)-2-methyl-3-(methylamino)-4-oxobutan-2-yl)benzoate (21.0 mg). LC-MS: 602 (M+H) + (1.47 min, measurement condition F)

[0398] b) Preparation of (S,E)-4-((S)-2-((S)-3-(4-(tert-butoxycarbonyl)phenyl)-3-methyl-2-(methoxyamino)butanamido)-N,3,3-trimethylbutanamido)-2,5-dimethylhex-2-enoic acid (Reference Example 24) tert-Butyl 4-((S)-4-(((S)-1-(((S,E)-6-ethoxy-2,5-dimethyl-6-oxohex-4-en-3-yl)(methyl)amino)-3,3-dimethyl-1-oxobutan-2-yl)amino)-2-methyl-3-(methylamino)-4-oxobutan-2-yl)benzoate (10.5 mg), methanol (3 mL) and water (1.000 mL) were mixed and added with lithium hydroxide (4.39 mg), and stirred at room temperature for 2 days. The solvent was distilled off under reduced pressure, and the residue was purified by reverse phase HPLC (mobile phase was 0.1% TFA water / 0.035% TFA acetonitrile solvent) to obtain Reference Example 24 (7.0 mg). LC-MS: 574 (M+H) + (1.49 min, measurement condition F)

[0399] Reference example 25 4-((S)-4-(((S)-1-(((S,E)-5-carboxy-2-methylhex-4-en-3-yl)(methyl)amino)-3,3-dimethyl-1-oxobutan-2-yl)amino)-2-methyl-3-(methylamino)-4-oxobutan-2-yl)benzoic acid [ka]

[0400] tert-Butyl 4-((S)-4-(((S)-1-(((S,E)-6-ethoxy-2,5-dimethyl-6-oxohex-4-en-3-yl)(methyl)amino)-3,3-dimethyl-1-oxobutan-2-yl)amino)-2-methyl-3-(methylamino)-4-oxobutan-2-yl)benzoate (10.5 mg), methanol (3 mL) and water (1 mL) were mixed and added with lithium hydroxide (4.39 mg), stirred at room temperature for 5 days, and then the solvent was distilled off under reduced pressure. The residue was dissolved in chloroform (4 mL), trifluoroacetic acid (1 mL) was added, stirred at room temperature for 17 hours, and then the solvent was distilled off under reduced pressure. The residue was purified by reverse phase HPLC (mobile phase was 0.1% TFA water / 0.035% TFA acetonitrile solvent) to obtain Reference Example 25 (7.40 mg). LC-MS: 518 (M+H) + (1.08 min, measurement condition F)

[0401] Reference example 26 (4S,E)-4-((2S)-2-(3-(4-hydroxyphenyl)-3-methyl-2-(methylamino)butyl)-N,3,3-trimethylbutanamido)-2,5-dimethylhex-2-enoic acid [ka]

[0402] a) Preparation of 3-(4-hydroxyphenyl)-3-methyl-2-(methylamino)butanoic acid Under a nitrogen atmosphere, a solution of 3-(4-hydroxyphenyl)-3-methyl-2-oxobutanoic acid (54.9 g) in anhydrous tetrahydrofuran (480 mL) was ice-cooled, and methylamine (280 mL) (2 mol / L tetrahydrofuran solution) was added dropwise. After stirring at room temperature for 1 hour, borane-pyridine complex (27.5 mL) was added dropwise, and the mixture was stirred at 55°C for 2.5 hours. Methanol (240 mL) was added dropwise under ice cooling, and the mixture was stirred at room temperature for 2 hours. After the solvent was distilled off under reduced pressure, tetrahydrofuran was added, and the suspension was suction-filtered. The powder was washed with tetrahydrofuran to obtain 3-(4-hydroxyphenyl)-3-methyl-2-(methylamino)butanoic acid (40.7 g). 1 H-NMR (400 MHz, D2O):1.21(3H, s), 1.24 (3H, s), 2.04 (3H, s), 3.06 (1H, s), 6.52 (2H, d, J = 8.8 Hz),7.11 (2H, d, J = 8.8).

[0403] b) Preparation of 2-((tert-butoxycarbonyl)(methyl)amino)-3-(4-((tert-butoxycarbonyl)oxy)phenyl)-3-methylbutanoic acid Under a nitrogen atmosphere, di-tert-butyl carbonate (39.9 g) and potassium carbonate (25.4 g) were added to a suspension of compound i1 (10.2 g) in 1,4-dioxane / water (1:1) (160 mL), and the mixture was stirred at 40° C. overnight. Ethyl acetate and water were added to the reaction solution, and the pH was adjusted to 2-3 with a 1 mol / L aqueous potassium hydrogen sulfate solution, followed by extraction with ethyl acetate. The organic layer was washed with saturated saline, dried over anhydrous magnesium sulfate, and the solvent was distilled off under reduced pressure. The residue was purified by silica gel chromatography (elution solvent: chloroform:methanol) to obtain 2-((tert-butoxycarbonyl)(methyl)amino)-3-(4-((tert-butoxycarbonyl)oxy)phenyl)-3-methylbutanoic acid. 1H-NMR (400 MHz, DMSO-D6):1.38(12H, s), 1.48 (12H, s), 2.64 (3H, s), 7.09 (2H, d, J = 8.4 Hz), 7.40 (2H, d, J= 8.8).

[0404] c) Preparation of (S)-2-((tert-butoxycarbonyl)(methyl)amino)-3-(4-hydroxyphenyl)-3-methylbutanoic acid Under a nitrogen atmosphere, compound i2 (15.7 g) was dissolved in dichloromethane (370 mL), and 28% sodium methoxide methanol solution (15.8 g) and methanol (14 mL) were added, followed by stirring at room temperature for 1.5 hours. Ethyl acetate and 4% potassium hydrogen sulfate aqueous solution were added to the reaction solution for extraction. The organic layer was washed with saturated saline, dried over anhydrous magnesium sulfate, and the solvent was distilled off under reduced pressure. The residue was purified by silica gel chromatography (elution solvent: chloroform:methanol) to obtain (S)-2-((tert-butoxycarbonyl)(methyl)amino)-3-(4-hydroxyphenyl)-3-methylbutanoic acid (153.7 mg). 1 H-NMR (400 MHz, DMSO-D6):1.36(12H, s), 1.42 (3H, s), 2.60 (3H, s), 6.66 (2H, d, J = 8.0 Hz), 7.15 (2H, d, J= 8.4).

[0405] d) Preparation of ethyl (9S,12S,E)-9-(tert-butyl)-6-(2-(4-hydroxyphenyl)propan-2-yl)-12-isopropyl-2,2,5,11,14-pentamethyl-4,7,10-trioxo-3-oxa-5,8,11-triazapentadec-13-en-15-oate A suspension of (S)-2-((tert-butoxycarbonyl)(methyl)amino)-3-(4-hydroxyphenyl)-3-methylbutanoic acid (153.7 mg), ethyl (S,E)-4-((S)-2-amino-N,3,3-trimethylbutanamido)-2,5-dimethylhex-2-enoate hydrochloride (117.6 mg), N-ethyl-N-isopropylpropan-2-amine (0.172 mL), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (129 mg), 1-hydroxybenzotriazole (103 mg) and N,N-dimethylformamide (5 mL) was stirred at room temperature for 17 hours. After the solvent was distilled off under reduced pressure, chloroform was added, the organic layer was washed with a saturated aqueous solution of sodium bicarbonate, dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The residue was purified by silica gel chromatography (eluent: hexane:ethyl acetate) to give ethyl (9S,12S,E)-9-(tert-butyl)-6-(2-(4-hydroxyphenyl)propan-2-yl)-12-isopropyl-2,2,5,11,14-pentamethyl-4,7,10-trioxo-3-oxa-5,8,11-triazapentadec-13-en-15-oate (206.3 mg). LC-MS: 618 (M+H) + (1.69 min, measurement condition F)

[0406] e) Preparation of ethyl (4S,E)-4-((2S)-2-(3-(4-hydroxyphenyl)-3-methyl-2-(methylamino)butanamido)-N,3,3-trimethylbutanamido)-2,5-dimethylhex-2-enoate Ethyl (9S,12S,E)-9-(tert-butyl)-6-(2-(4-hydroxyphenyl)propan-2-yl)-12-isopropyl-2,2,5,11,14-pentamethyl-4,7,10-trioxo-3-oxa-5,8,11-triazapentadec-13-en-15-oate (189.2 mg) and chloroform (4 mL) were added to the mixture, stirred at room temperature for 1 hour, and the solvent was distilled off under reduced pressure. Chloroform was added to the residue, the organic layer was washed with a saturated aqueous solution of sodium bicarbonate, dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The residue was purified by silica gel chromatography (eluent: hexane:ethyl acetate) to give ethyl (4S,E)-4-((2S)-2-(3-(4-hydroxyphenyl)-3-methyl-2-(methylamino)butanamido)-N,3,3-trimethylbutanamido)-2,5-dimethylhex-2-enoate (110.1 mg). LC-MS: 518 (M+H) + (1.09 min, measurement condition F)

[0407] f) Preparation of (4S,E)-4-((2S)-2-(3-(4-hydroxyphenyl)-3-methyl-2-(methylamino)butyl)-N,3,3-trimethylbutanamido)-2,5-dimethylhex-2-enoic acid (Reference Example 26) Ethyl (4S,E)-4-((2S)-2-(3-(4-hydroxyphenyl)-3-methyl-2-(methylamino)butanamido)-N,3,3-trimethylbutanamido)-2,5-dimethylhex-2-enoate (110.1 mg), methanol (3 mL) and water (1 mL) were mixed, and lithium hydroxide (35.7 mg) was added under ice cooling and stirred at room temperature for 2 days. The solvent was distilled off under reduced pressure, and the residue was purified by reverse phase HPLC (mobile phase: 0.1% TFA water / 0.035% TFA acetonitrile solvent) to obtain Reference Example 26 (113.2 mg). LC-MS: 490 (M+H) + (1.03 min, measurement condition F)

[0408] Reference example 27 ((4S,E)-4-((2S)-2-(3-(4-hydroxyphenyl)-3-methyl-2-(methylamino)butanamido)-N,3,3-trimethylbutanamido)-2,5-dimethylhex-2-enoyl)-D-glutamic acid [ka]

[0409] a) Preparation of dimethyl ((4S,E)-4-((2S)-2-(3-(4-hydroxyphenyl)-3-methyl-2-(methylamino)butanamido)-N,3,3-trimethylbutanamido)-2,5-dimethylhex-2-enoyl)-D-glutamic acid A mixture of (4S,E)-4-((2S)-2-(3-(4-hydroxyphenyl)-3-methyl-2-(methylamino)butanamido)-N,3,3-trimethylbutanamido)-2,5-dimethylhex-2-enoic acid trifluoroacetate (21.4 mg), N-ethyl-N-isopropylpropan-2-amine (22.91 mg), dimethyl D-glutamic acid hydrochloride (15.01 mg), 3-(((ethylimino)methylene)amino)-N,N-dimethylpropan-1-amine hydrochloride (13.59 mg), 1-hydroxybenzotriazole (9.58 mgl) and N,N-dimethylformamide (1 mL) was stirred at room temperature for 15 hours. Ethyl acetate was added to the reaction solution, and the mixture was washed with saturated saline, after which the organic layer was dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The residue was purified by silica gel chromatography (eluent: chloroform / methanol) to give dimethyl ((4S,E)-4-((2S)-2-(3-(4-hydroxyphenyl)-3-methyl-2-(methylamino)butanamido)-N,3,3-trimethylbutanamido)-2,5-dimethylhex-2-enoyl)-D-glutamate (21.6 mg). LC-MS: 647 (M+H) + (1.21min, measurement condition F)

[0410] b) Preparation of ((4S,E)-4-((2S)-2-(3-(4-hydroxyphenyl)-3-methyl-2-(methylamino)butanamido)-N,3,3-trimethylbutanamido)-2,5-dimethylhex-2-enoyl)-D-glutamic acid (Reference Example 27) Dimethyl ((4S,E)-4-((2S)-2-(3-(4-hydroxyphenyl)-3-methyl-2-(methylamino)butanamido)-N,3,3-trimethylbutanamido)-2,5-dimethylhex-2-enoyl)-D-glutamic acid (21.6 mg), methanol (3 mL) and water (1 mL) were mixed and added with lithium hydroxide (5.61 mg) at room temperature and stirred. The solvent was distilled off under reduced pressure. The residue was purified by high performance liquid chromatography to obtain Reference Example 27 (15.8 mg). LC-MS: 619 (M+H) + (1.04 min, measurement condition F)

[0411] Reference example 28 N6-(tert-butoxycarbonyl)-N2-((R)-4-((S,E)-4-((S)-2-((S)-2-((tert-butoxycarbonyl)(methyl)amino)-3-(5-fluoro-1-methyl-1H-indol-3-yl)-3-methylbutanamido)-N,3,3-trimethylbutanamido)-2,5-dimethylhex-2-enamido)-4-carboxybutanoyl)lysine [ka]

[0412] a) Preparation of methyl N6-(tert-butoxycarbonyl)-N2-((R)-4-((S,E)-4-((S)-2-((S)-2-((tert-butoxycarbonyl)(methyl)amino)-3-(5-fluoro-1-methyl-1H-indol-3-yl)-3-methylbutanamido)-N,3,3-trimethylbutanamido)-2,5-dimethylhex-2-enamido)-5-ethoxy-5-oxopentanoyl) lysinate (6S,9S,12S,17R,E)-9-(tert-butyl)-17-(ethoxycarbonyl)-6-(2-(5-fluoro-1-methyl-1H-indol-3-yl)propan-2-yl)-12-isopropyl-2,2,5,11,14-pentamethyl-4,7,10,15-tetraoxo-3-oxa-5,8,11,16-tetraazaicos-13-en-20-oic acid (54.5 mg) and N,N-dimethylformamide (2 mL) were added to a mixture of the above under ice cooling, and the mixture was stirred for 30 minutes. N-(tert-butoxycarbonyl)-L-lysine methyl ester hydrochloride (24.20 mg) and N-ethyl-N-isopropylpropan-2-amine (0.028 mL) were added and stirred at room temperature for 17 hours, after which the solvent was distilled off under reduced pressure. The residue was purified by silica gel chromatography (elution solvent: chloroform:methanol) to obtain methyl N6-(tert-butoxycarbonyl)-N2-((R)-4-((S,E)-4-((S)-2-((S)-2-((tert-butoxycarbonyl)(methyl)amino)-3-(5-fluoro-1-methyl-1H-indol-3-yl)-3-methylbutanamide)-N,3,3-trimethylbutanamide)-2,5-dimethylhex-2-enamide)-5-ethoxy-5-oxopentanoyl) lysineate (71 mg). LC-MS: 1067 (M+Na) + (1.557 min, measurement condition G)

[0413] b) Preparation of N6-(tert-butoxycarbonyl)-N2-((R)-4-((S,E)-4-((S)-2-((S)-2-((tert-butoxycarbonyl)(methyl)amino)-3-(5-fluoro-1-methyl-1H-indol-3-yl)-3-methylbutanamido)-N,3,3-trimethylbutanamido)-2,5-dimethylhex-2-enamido)-4-carboxybutanoyl)lysine (Reference Example 28) A mixture of methyl N6-(tert-butoxycarbonyl)-N2-((R)-4-((S,E)-4-((S)-2-((S)-2-((tert-butoxycarbonyl)(methyl)amino)-3-(5-fluoro-1-methyl-1H-indol-3-yl)-3-methylbutanamido)-N,3,3-trimethylbutanamido)-2,5-dimethylhex-2-enamido)-5-ethoxy-5-oxopentanoyl) lysinate (71 mg), methanol (4 mL) and 1 mol / L lithium hydroxide aqueous solution (4 mL) was stirred at 60° C. for 17 hours. The reaction solution was acidified by adding a saturated citric acid aqueous solution, and then extracted with ethyl acetate. The organic layer was dried over anhydrous magnesium sulfate, and the solvent was distilled off under reduced pressure. The residue was purified by ODS column chromatography (elution solvent: water:acetonitrile) to obtain Reference Example 28 (49.0 mg). LC-MS: 1024 (M+Na) + (1.416min, measurement condition G)

[0414] Reference example 29 Di-tert-butyl ((S,E)-4-((S)-2-((R)-1-(tert-butoxycarbonyl)piperidine-2-carboxamido)-N,3,3-trimethylbutanamido)-2,5-dimethylhex-2-enoyl)-D-glutamate [ka]

[0415] a) Preparation of (R)-1-(tert-butoxycarbonyl)piperidine-2-carboxylic acid A mixture of D-pipecolic acid, di-tert-butyl dicarbonate (8.36 mL), 5 mol / L aqueous sodium hydroxide solution (19.20 mL), tetrahydrofuran (10 mL) and water (10 mL) was stirred at room temperature for 8 hours, and the solvent was removed under reduced pressure. The aqueous phase was washed with diethyl ether, neutralized (pH 7) with 0.1 mol / L aqueous hydrochloric acid, and extracted with diethyl ether. The mixture was dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to give (R)-1-(tert-butoxycarbonyl)piperidine-2-carboxylic acid (432.3 mg). LC-MS: 228(MH) - (1.51min, measurement condition F)

[0416] b) Preparation of tert-butyl (R)-2-(((S)-1-(((S,E)-6-ethoxy-2,5-dimethyl-6-oxohex-4-en-3-yl)(methyl)amino)-3,3-dimethyl-1-oxobutan-2-yl)carbamoyl)piperidine-1-carboxylate A suspension of (R)-1-(tert-butoxycarbonyl)piperidine-2-carboxylic acid (432.3 mg), ethyl (S,E)-4-((S)-2-amino-N,3,3-trimethylbutanamido)-2,5-dimethylhex-2-enoate (393 mg), o-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (953 mg), N-ethyl-N-isopropylpropan-2-amine (0.659 mL) and N,N-dimethylformamide (3 mL) was stirred at room temperature for 15 hours, and then extracted with ethyl acetate. The organic layer was washed with a saturated aqueous solution of sodium bicarbonate and saturated saline, and then dried over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure. The residue was purified by silica gel chromatography (eluent: hexane:ethyl acetate) to give tert-butyl (R)-2-(((S)-1-(((S,E)-6-ethoxy-2,5-dimethyl-6-oxohex-4-en-3-yl)(methyl)amino)-3,3-dimethyl-1-oxobutan-2-yl)carbamoyl)piperidine-1-carboxylate (658 mg). LC-MS: 546 (M+Na)+ (1.51min, measurement condition F)

[0417] c) Preparation of (((S,E)-4-((S)-2-((R)-1-(tert-butoxycarbonyl)piperidine-2-carboxamide)-N,3,3-trimethylbutanamide)-2,5-dimethylhex-2-enoic acid A mixture of tert-butyl (R)-2-(((S)-1-(((S,E)-6-ethoxy-2,5-dimethyl-6-oxohex-4-en-3-yl)(methyl)amino)-3,3-dimethyl-1-oxobutan-2-yl)carbamoyl)piperidine-1-carboxylate (508.1 mg), methanol (8 mL), water (2 mL) and lithium hydroxide (204 mg) was stirred at room temperature for 4 days, and then extracted with ethyl acetate. The organic layer was washed with saturated saline and then dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure to obtain (((S,E)-4-((S)-2-((R)-1-(tert-butoxycarbonyl)piperidine-2-carboxamide)-N,3,3-trimethylbutanamide)-2,5-dimethylhex-2-enoic acid. The obtained compound was used in the next reaction as it was without purification. LC-MS: 518 (M+Na) + (1.19 min, measurement condition F)

[0418] d) Preparation of di-tert-butyl ((S,E)-4-((S)-2-((R)-1-(tert-butoxycarbonyl)piperidine-2-carboxamido)-N,3,3-trimethylbutanamido)-2,5-dimethylhex-2-enoyl)-D-glutamate (Reference Example 29) A mixture of (S,E)-4-((S)-2-((R)-1-(tert-butoxycarbonyl)piperidine-2-carboxamide)-N,3,3-trimethylbutanamide)-2,5-dimethylhex-2-enoic acid (481 mg), N-ethyl-N-isopropylpropan-2-amine (0.501 mL), 3-(((ethylamino)methylene)amino)-N,N-dimethylpropan-1-amine hydrochloride (372 mg), 1H-benzo[d][1,2,3]triazol-1-ol (262 mg), D-glutamic acid di-tert-butyl ester hydrochloride (287 mg) and N,N-dimethylformamide (10 mL) was stirred at room temperature for 20 hours, and then extracted with ethyl acetate. The organic layer was washed with a saturated aqueous solution of sodium bicarbonate and saturated saline, and then dried over anhydrous sodium sulfate. The residue was purified by silica gel chromatography (eluent: hexane:ethyl acetate) to give Reference Example 29 (673.0 mg). LC-MS: 759 (M+Na) + (1.35min, measurement condition H)

[0419] Reference examples 30~52 Intermediate A15 of Reference Example 1, Reference Example 2 and Reference Example 53 were used as raw materials, and reacted and treated in the same manner as in step p) of Reference Example 1 to give the compounds shown in Table 2 below. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5]

[0420] Reference example 53 The corresponding starting compounds were used and reacted and treated in the same manner as in Reference Example 1 to obtain the compounds shown in Table 3 below. [Table 3]

[0421] Reference examples 54~73 The corresponding starting compounds were used and treated in the same manner as in step c) of Reference Example 3 to give the compounds shown in Table 4 below.

[0422] [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4]

[0423] Reference examples 74~78 The corresponding starting compounds were used and treated in the same manner as in Example M1 to give the compounds shown in Table 5 below. [Table 5]

[0424] Reference examples 79~88 The corresponding starting compounds and Reference Example 5 were used for reaction and treatment in the same manner as in step m) of Reference Example 1 to give the compounds shown in Table 6 below. [Table 6-1] [Table 6-2]

[0425] Reference examples 89~95 The corresponding starting compounds were used in the same manner as in step o) of Reference Example 1 to give the compounds shown in Table 7 below. [Table 7]

[0426] Reference example 96 The corresponding starting compounds were used and reacted and treated in the same manner as in Example M1 to obtain the compounds shown in Table 8 below. [Table 8]

[0427] Reference example 97 (R)-4-((R)-4-((R)-4-amino-5-(tert-butoxy)-5-oxopentanamide)-5-(tert-butoxy)-5-oxopentanamide)-5-(tert-butoxy)-5-oxopentanoic acid [ka]

[0428] a) Preparation of 1-(tert-butyl) 5-(2,5-dioxopyrrolidin-1-yl) (((9H-fluoren-9-yl)methoxy)carbonyl)-D-glutamic acid In the same manner as in Reference Example 2-c), 1-(tert-butyl) 5-(2,5-dioxopyrrolidin-1-yl) (((9H-fluoren-9-yl)methoxy)carbonyl)-D-glutamic acid (520 mg) was obtained from (R)-4-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(tert-butoxy)-5-oxopentanoic acid (500 mg). LC-MS: 523 (M+H) + (1.324min, measurement condition G)

[0429] b) Preparation of (R)-4-((R)-4-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(tert-butoxy)-5-oxopentanamide)-5-(tert-butoxy)-5-oxopentanoic acid Using a method similar to that used in Reference Example 3, (R)-4-((R)-4-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(tert-butoxy)-5-oxopentanamide)-5-(tert-butoxy)-5-oxopentanoic acid (428.7 mg) was obtained from 1-(tert-butyl) 5-(2,5-dioxopyrrolidin-1-yl) (((9H-fluoren-9-yl)methoxy)carbonyl)-D-glutamic acid (520 mg). LC-MS: 611 (M+H) + (1.551min, measurement condition G)

[0430] c) Preparation of 1-(tert-butyl) 5-(2,5-dioxopyrrolidin-1-yl)((R)-4-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(tert-butoxy)-5-oxopentanoyl)-D-glutamic acid Using a method similar to that used in Reference Example 2-c), 1-(tert-butyl) 5-(2,5-dioxopyrrolidin-1-yl)((R)-4-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(tert-butoxy)-5-oxopentanoyl)-D-glutamic acid (33 mg) was obtained from (R)-4-((R)-4-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(tert-butoxy)-5-oxopentanamide)-5-(tert-butoxy)-5-oxopentanoic acid (100 mg). LC-MS: 708 (M+H) + (1.598min, measurement condition G)

[0431] d) Preparation of (5R,10R,15R)-5,10,15-tris(tert-butoxycarbonyl)-1-(9H-fluoren-9-yl)-3,8,13-trioxo-2-oxa-4,9,14-triazaoctadecane-18-oic acid Using the same method as in Reference Example 3, (5R,10R,15R)-5,10,15-tris(tert-butoxycarbonyl)-1-(9H-fluoren-9-yl)-3,8,13-trioxo-2-oxa-4,9,14-triazaoctadecane-18-oic acid (16.6 mg) was obtained from 1-(tert-butyl) 5-(2,5-dioxopyrrolidin-1-yl)((R)-4-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(tert-butoxy)-5-oxopentanoyl)-D-glutamic acid (33 mg). LC-MS: 796 (M+H) + (1.119 min, measurement condition F)

[0432] e) Preparation of (R)-4-((R)-4-((R)-4-amino-5-(tert-butoxy)-5-oxopentanamide)-5-(tert-butoxy)-5-oxopentanamide)-5-(tert-butoxy)-5-oxopentanoic acid (Reference Example 97) In the same manner as in Reference Example 103-b), Reference Example 97 (7.5 mg) was obtained from (5R,10R,15R)-5,10,15-tris(tert-butoxycarbonyl)-1-(9H-fluoren-9-yl)-3,8,13-trioxo-2-oxa-4,9,14-triazaoctadecane-18-oic acid (16.6 mg). LC-MS: 574 (M+H) + (0.698min, measurement condition F)

[0433] Reference example 98 The corresponding starting compounds were used and treated in the same manner as in Reference Example 2 to obtain the compounds shown in Table 9 below. [Table 9]

[0434] Reference example 99 Using Reference Example 98 and Reference Example 5, reactions and treatments were carried out in the same manner as in step m) of Reference Example 1 to give the compounds shown in Table 10 below. [Table 10]

[0435] Reference example 100 Di-tert-butyl ((4S,E)-4-((2S)-2-(3-(4-((2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)carbamoyl)phenyl)-3-methyl-2-(methylamino)butanamido)-N,3,3-trimethylbutanamido)-2,5-dimethylhex-2-enoyl)-D-glutamic acid [ka]

[0436] a) Preparation of tert-butyl 4-(4-(benzyloxy)-3-((tert-butoxycarbonyl)(methyl)amino)-2-methyl-4-oxobutan-2-yl)benzoate Benzyl bromide (0.647 mL) was added to a suspension of 2-((tert-butoxycarbonyl)(methyl)amino)-3-(4-(tert-butoxycarbonyl)phenyl)-3-methylbutanoic acid (1.48 g), sodium carbonate (0.77 g) and N,N-dimethylformamide (7 mL), and the mixture was stirred at room temperature for 17 hours. Ethyl acetate was added to the reaction solution. The organic layer was washed with water and saturated saline, then dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The residue was purified by silica gel chromatography (elution solvent: hexane:ethyl acetate) to give tert-butyl 4-(4-(benzyloxy)-3-((tert-butoxycarbonyl)(methyl)amino)-2-methyl-4-oxobutan-2-yl)benzoate (1.78 g). LC-MS: 520 (M+Na) + (1.778min, measurement condition G)

[0437] b) Preparation of 4-(4-(benzyloxy)-2-methyl-3-(methylamino)-4-oxobutan-2-yl)benzoic acid trifluoroacetate Trifluoroacetic acid (10 mL) was added to a mixture of tert-butyl 4-(4-(benzyloxy)-3-((tert-butoxycarbonyl)(methyl)amino)-2-methyl-4-oxobutan-2-yl)benzoate (1.78 g) and chloroform (40 mL), and the mixture was stirred at room temperature for 5 hours. The solvent was distilled off under reduced pressure to obtain 4-(4-(benzyloxy)-2-methyl-3-(methylamino)-4-oxobutan-2-yl)benzoic acid trifluoroacetate. The compound was used in the next reaction without purification. LC-MS: 342 (M+H) + (1.05min, measurement condition F)

[0438] c) Preparation of methyl 4-(4-(benzyloxy)-2-methyl-3-(methylamino)-4-oxobutan-2-yl)benzoate Iodomethane (0.169 mL) was added to a suspension of 4-(4-(benzyloxy)-2-methyl-3-(methylamino)-4-oxobutan-2-yl)benzoic acid trifluoroacetate, sodium carbonate (379 mg) and N,N-dimethylformamide (9 mL) and stirred at room temperature. Ethyl acetate was added to the reaction solution. The organic layer was washed with water and saturated saline, then dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The residue was purified by silica gel chromatography (elution solvent: hexane:ethyl acetate) to give methyl 4-(4-(benzyloxy)-2-methyl-3-(methylamino)-4-oxobutan-2-yl)benzoate (362.3 mg). LC-MS: 356 (M+H) + (1.08 min, measurement conditions)

[0439] d) Preparation of 3-(4-(methoxycarbonyl)phenyl)-3-methyl-2-(methylamino)butanoic acid A suspension of methyl 4-(4-(benzyloxy)-2-methyl-3-(methylamino)-4-oxobutan-2-yl)benzoate (362.3 mg), palladium-carbon (85.5 mg) and ethyl acetate (10 mL) was stirred at room temperature for 5 hours under a hydrogen atmosphere. The mixture was further stirred for 5 hours. The reaction solution was filtered through filter paper, and the solvent was distilled off under reduced pressure to obtain 3-(4-(methoxycarbonyl)phenyl)-3-methyl-2-(methylamino)butanoic acid. LC-MS: 266 (M+H) + (0.82 min, measurement condition F)

[0440] e) Preparation of di-tert-butyl ((S,E)-4-((S)-2-((tert-butoxycarbonyl)amino)-N,3,3-trimethylbutanamido)-2,5-dimethylhex-2-enoyl)-D-glutamic acid A mixture of (S,E)-4-((S)-2-((tert-butoxycarbonyl)amino)-N,3,3-trimethylbutanamido)-2,5-dimethylhex-2-enoic acid (220.2 mg), di-tert-butyl D-glutamic acid hydrochloride (254 mg), N-ethyl-N-isopropylpropan-2-amine (0.300 mL), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (220 mg), 1-hydroxybenzotriazole (175 mg) and N,N-dimethylformamide was stirred at room temperature for 17 hours. Ethyl acetate was added to the reaction solution. The organic layer was washed with water and saturated saline, then dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. Di-tert-butyl ((S,E)-4-((S)-2-((tert-butoxycarbonyl)amino)-N,3,3-trimethylbutanamido)-2,5-dimethylhex-2-enoyl)-D-glutamic acid (232.4 mg) was obtained. LC-MS: 626 (M+H) + (1.76min, measurement condition F)

[0441] f) Preparation of di-tert-butyl ((S,E)-4-((S)-2-amino-N,3,3-trimethylbutanamido)-2,5-dimethylhex-2-enoyl)-D-glutamic acid A solution of hydrochloric acid (13.54 mg) in ethyl acetate was added to a mixture of di-tert-butyl ((S,E)-4-((S)-2-((tert-butoxycarbonyl)amino)-N,3,3-trimethylbutanamido)-2,5-dimethylhex-2-enoyl)-D-glutamic acid (232.4 mg) and ethyl acetate (3.7 mL) under ice cooling, and the mixture was stirred at room temperature for 1 hour and 20 minutes. The reaction solution was ice-cooled, 28% aqueous ammonia was added, and the mixture was extracted with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure to obtain di-tert-butyl ((S,E)-4-((S)-2-amino-N,3,3-trimethylbutanamido)-2,5-dimethylhex-2-enoyl)-D-glutamic acid (62.2 mg). The compound was used in the next reaction without purification. LC-MS: 526 (M+H) + (1.14 min, measurement condition F)

[0442] g) Preparation of di-tert-butyl ((4S,E)-4-((2S)-2-(3-(4-(methoxycarbonyl)phenyl)-3-methyl-2-(methylamino)butanamido)-N,3,3-trimethylbutanamido)-2,5-dimethylhex-2-enoyl)-D-glutamic acid A suspension of di-tert-butyl ((S,E)-4-((S)-2-amino-N,3,3-trimethylbutanamido)-2,5-dimethylhex-2-enoyl)-D-glutamic acid (31.4 mg), di-tert-butyl ((S,E)-4-((S)-2-amino-N,3,3-trimethylbutanamido)-2,5-dimethylhex-2-enoyl)-D-glutamic acid (62.2 mg), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (45.4 mg), 1-hydroxybenzotriazole (32.0 mg), N-ethyl-N-isopropylpropan-2-amine (0.062 mL) and N,N-dimethylformamide (2 mL) was stirred at room temperature for 17 hours. The solvent was distilled off under reduced pressure. Ethyl acetate was added to the residue, which was washed with water, saturated aqueous sodium bicarbonate solution, and saturated saline, and the organic layer was dried over anhydrous sodium sulfate and the solvent was distilled off under reduced pressure. The residue was purified by silica gel chromatography (elution solvent: hexane:ethyl acetate) to give di-tert-butyl ((4S,E)-4-((2S)-2-(3-(4-(methoxycarbonyl)phenyl)-3-methyl-2-(methylamino)butanamido)-N,3,3-trimethylbutanamido)-2,5-dimethylhex-2-enoyl)-D-glutamic acid (60.1 mg). LC-MS: 774 (M+H) + (1.341min, measurement condition G)

[0443] h) Preparation of 4-((7R,12S,15S,E)-7-(tert-butoxycarbonyl)-15-(tert-butyl)-12-isopropyl-2,2,10,13,19-pentamethyl-18-(methylamino)-4,9,14,17-tetraoxo-3-oxa-8,13,16-triazaycos-10-en-19-yl)benzoic acid To a mixture of tert-butyl ((4S,E)-4-((2S)-2-(3-(4-(methoxycarbonyl)phenyl)-3-methyl-2-(methylamino)butanamido)-N,3,3-trimethylbutanamido)-2,5-dimethylhex-2-enoyl)-D-glutamate (56.3 mg), methanol (4 mL) and water (1 mL), lithium hydroxide (9.17 mg) was added under ice cooling and stirred at room temperature for 17 hours. The solvent was distilled off under reduced pressure. The residue was purified by reverse phase HPLC (mobile phase: 0.1% TFA water / 0.035% TFA acetonitrile solvent) to give 4-((7R,12S,15S,E)-7-(tert-butoxycarbonyl)-15-(tert-butyl)-12-isopropyl-2,2,10,13,19-pentamethyl-18-(methylamino)-4,9,14,17-tetraoxo-3-oxa-8,13,16-triazaycos-10-en-19-yl)benzoic acid (14.8 mg). LC-MS: 759 (M+H) + (1.334min, measurement condition G)

[0444] i) Preparation of di-tert-butyl ((4S,E)-4-((2S)-2-(3-(4-((2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)carbamoyl)phenyl)-3-methyl-2-(methylamino)butanamido)-N,3,3-trimethylbutanamido)-2,5-dimethylhex-2-enoyl)-D-glutamic acid (Reference Example 100) 4-((7R,12S,15S,E)-7-(tert-butoxycarbonyl)-15-(tert-butyl)-12-isopropyl-2,2,10,13,19-pentamethyl-18-(methylamino)-4,9,14,17-tetraoxo-3-oxa-8,13,16-triazaicos-10-en-19-yl)benzoic acid (14.8 mg), 3-(((ethylimino)methylene)amino)-N,N-dimethylpropan-1-amine hydrochloride (7.48 mg), 1-hydroxybenzotriazole (5.97 mg), N-ethyl-N-isopropylpropan-2-amine (0.014 mL), 1-(2-aminoethyl)-pyrrole-2,5-dione A mixture of hydrochloride (6.86 mg) and N,N-dimethylformamide (2 mL) was stirred at room temperature for 3 hours. Ethyl acetate was added, and the mixture was washed with saturated aqueous sodium bicarbonate and saturated saline. The organic layer was dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The residue was purified by silica gel chromatography (elution solvent: chloroform:methanol) to obtain Reference Example 100 (9.00 mg). LC-MS: 881 (M+H) + (1.287min, measurement condition G)

[0445] Example M1 (3S,6S,9S,10E,14R)-6-tert-butyl-14-(3-{[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]amino}-3-oxopropyl)-8,11-dimethyl-3-[2-(1-methyl-1H-indol-3-yl)propan-2-yl]-4,7,12-trioxo-9-(propan-2-yl)-2,5,8,13-tetraazapentadec-10-ene-15-carboxylic acid [ka] Trifluoroacetic acid (0.2 mL) was added to a chloroform solution (1.0 mL) of Reference Example 1 (14 mg), and the mixture was stirred at 25° C. for 4 hours. Then, trifluoroacetic acid (0.2 mL) was added, and the mixture was stirred for another 2 hours at 25° C. After the reaction was completed, the solvent was distilled off under reduced pressure, and the residue was purified by reverse phase column chromatography (elution solvent: 0.1% TFA acetonitrile:water) to obtain Example M1 (2.8 mg). LC-MS: 778 (M+H) + , 776 (MH) - (1.081 min, measurement condition D)

[0446] Example M2 N-{(2E,4S)-2,5-dimethyl-4-[methyl(N,β,β,1-tetramethyl-L-tryptophyll-3-methyl-L-valyl)amino]hex-2-enoyl}-D-γ-glutamyl-N 6 -[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-lysine

[0447] [ka] A mixture of Reference Example 4 (7.2 mg), N-succinimidyl 6-maleimidohexanoate (2.8 mg), N,N-diisopropylethylamine (2.3 mg) and N,N-dimethylformamide (1 mL) was stirred for 18 hours at 25° C. After completion of the reaction, the reaction solution was purified by reverse phase column chromatography (elution solvent: 0.1% TFA acetonitrile:water) to obtain Example M2 (2.8 mg). LC-MS: 977 (M+H) + , 975 (MH) - (1.061 min, measurement condition D)

[0448] Examples M3 to M8 The corresponding starting compounds were reacted and treated in the same manner as in Example M1 or M2 to obtain the compounds shown in Table 11 below. nIn the formula shown in the column "A", "L" is (AA) n In addition, in the formula shown in the column "L" in Table 11, "(AA) n " is one of the two ends of L (AA) n The figure shows the end that is bound to [Table 11]

[0449] Examples M9 to M25 The corresponding starting compounds were used and treated in the same manner as in Example M1 to give the compounds shown in Table 12 below. [Table 12-1] [Table 12-2]

[0450] Examples M26 to M30 The corresponding starting compounds were used and treated in the same manner as in Example M2 to obtain the compounds shown in Table 13 below. [Table 13]

[0451] Examples M31 to M40 The corresponding starting compounds were used and treated in the same manner as in Example M1 to obtain the compounds shown in Table 14 below. [Table 14-1] [Table 14-2]

[0452] Examples M41 to M46 The corresponding starting compounds were used and treated in the same manner as in step p) of Reference Example 1 to give the compounds shown in Table 15 below. [Table 15]

[0453] Example M47 ((4S,E)-4-((2S)-2-(3-(4-((2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)carbamoyl)phenyl)-3-methyl-2-(methylamino)butanamido)-N,3,3-trimethylbutanamido)-2,5-dimethylhex-2-enoyl)-D-glutamic acid [ka] Trifluoroacetic acid (1 mL) was added to a mixture of Reference Example 100 (9.0 mg) and chloroform (4 mL), and the mixture was stirred at room temperature for 4 hours. The solvent was distilled off under reduced pressure. The residue was purified by reverse phase HPLC (mobile phase: 0.1% TFA water / 0.035% TFA acetonitrile solvent) to obtain Example M47 (5.4 mg). LC-MS: 769 (M+H) + (0.919min, measurement condition G)

[0454] Example M48 Reference Example 96 was used as a starting compound, and the reaction and treatment were carried out in the same manner as in Example M2 to obtain the compounds shown in Table 16 below.

[0455] [Table 16]

[0456] Example M49 N 2 -((S,E)-4-((S)-2-((S)-2-(dimethylamino)-3-methyl-3-phenylbutanamide)-N,3,3-trimethylbutanamide)-2,5-dimethylhex-2-enoyl)-N 5-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)ethyl)-D-glutamine [ka] To a solution of monotrifluoroacetate (10 mg) of Example M4 in acetonitrile (1 mL), an aqueous formaldehyde solution (1 mL) was added, and sodium triacetoxyborate (15 mg) was added and stirred at room temperature for 3 hours. The reaction solution was concentrated, and then purified by reverse phase HPLC (mobile phase: 0.1% TFA water / 0.035% TFA acetonitrile solvent) to obtain Example M49 (5.4 mg). LC-MS: 739 (M+H) + (1.007 min, measurement condition G)

[0457] Example M50 Reference Example 99 was used as a starting compound, and the reaction and treatment were carried out in the same manner as in Example M1 to obtain the compounds shown in the table below. [Table 17]

[0458] The NMR data for Examples M4, M29, M31, M32, M33, M35 and M41 to M45 are shown in the table below. [Table 18-1] [Table 18-2]

[0459] The antibodies used in the example ADCs are either commercially available or can be prepared according to the references shown in the table below. [Table 19]

[0460] Example ADC1 Brentuximab-Example M3 Conjugate (Average Drug-Antibody Ratio: 7.55) [ka] To a phosphate buffered saline solution (0.039 mL) of brentuximab (1 mg), 1 mmol / L tris(2-carboxyethyl)phosphine (TCEP) in trishydroxymethylaminomethane hydrochloride buffer (0.133 mL) was added, and the mixture was incubated at 37° C. for 45 minutes. The antibody solution was cooled to 0° C., and then treated with a PD-10 desalting column pre-equilibrated with phosphate buffered saline to obtain a phosphate buffered saline solution of reduced brentuximab. After cooling to 0° C., a 1 mmol / L DMSO solution (0.133 mL) of Example M3 diluted 10-fold with phosphate buffered saline was added, thoroughly mixed, and incubated at 4° C. for 16 hours. The mixture was then purified with a PD-10 desalting column pre-equilibrated with phosphate buffered saline, and centrifuged to obtain Example ADC1 (0.819 mg).

[0461] The average DAR of the obtained ADC was measured by reducing or non-reducing SDS-PAGE or HPLC-HIC. The average DAR can also be qualitatively or quantitatively measured by ultraviolet-visible absorption spectroscopy (UV-Vis), reducing or non-reducing SDS-PAGE, HPLC-HIC, SEC, RP-HPLC, LC-MS, etc. These techniques are described in Antibody Drug Conjugates, Methods in Molecular Biology vol.1045,2013.pp267-284.L.Ducry,Ed.

[0462] As a result of HPLC-HIC analysis (measurement condition E) of Example ADC1 obtained by the above protocol, the Rt of the peak of Example ADC1 having a DAR of 8 was 5.67 min.

[0463] Examples ADC2 to ADC9 The corresponding antibody and modifying agent (the compound of the Example) were reacted and treated in the same manner as in Example ADC1 to obtain the ADC shown in Table 20 below. [ka] [Table 20]

[0464] The Rt(min) of the ADC of the Example in Table 20 above is that of the peak of the ADC with a DAR of 8, as observed by HPLC-HIC analysis (measurement condition E). The Rt(min) of the ADC of Comparative Example 3 is that of the peak of the ADC with a DAR of 8.

[0465] The comparative example compounds in Table 20 above refer to the following compounds disclosed in International Publication No. 2014 / 057436 (Patent Document 8). [ka]

[0466] Examples ADC10 to 44 Brentuximab and a modifying agent (an example compound) were reacted and treated in the same manner as in Example ADC1 to obtain an Example ADC shown in Table 21 below. [Table 21]

[0467] "ADC production was confirmed by SDS-PAGE" means that, as a result of SDS-PAGE analysis of the ADC of the example under disulfide non-reducing conditions using SeeBlue (registered trademark) Plus2 (Thermo Fisher Scientific) as a marker, strong bands were detected at molecular weights of about 50 kDa and 25 kDa. This indicates that the modifying agent was conjugated to cysteine ​​residues involved in the disulfide bonds between the antibody light chain and heavy chain and in the hinge, and that an ADC was obtained.

[0468] Examples ADC45-62 The example ADCs shown in Table 22 below were obtained in the same manner as in Example ADC1 using antibodies and modifying agents (compounds of the examples). [Table 22]

[0469] Examples ADC63 to ADC64 Brentuximab and a modifying agent (an example compound) were used and reacted and treated in the same manner as in Example ADC1 to obtain the example ADC shown in Table 23 below. [Table 23]

[0470] Test Example The pharmacological test results of specific examples of the antibody-drug conjugate of the present invention are shown below, and their pharmacological actions are explained, but the present invention is not limited to these test examples.

[0471] Test Example 1: Cytotoxicity test (1) Karpas-299 cells (European Collection of Authenticated Cell Cultures, hereafter referred to as ECACC), a human lymphoma cell line, were cultured in RPMI1640 (GIBCO) (hereafter referred to as "medium") containing 10% fetal bovine serum (MP Biomedicals). The cells were cultured at 2 × 10 6The concentration was adjusted to 1000 cells / mL, and 50 μL of each was added to a 96-well cell culture microplate. 50 μL of the compound of the Example or the Comparative Example compound, diluted 4-fold in 8 steps with the medium, was added to the microplate. These were cultured at 37°C and 5% CO2 for 4 days. After culture, the microplate was removed from the incubator and allowed to stand at room temperature for 10 minutes. 50 μL of CellTiter-Glo Luminescent Cell Viability Assay (Promega) was added to each well and stirred. This mixture was incubated in the dark for 20 minutes. The cell viability was calculated by measuring the luminescence in each well using a microplate luminometer. IC 50 The values ​​were calculated and the results are shown in Table 24.

[0472] I C 50 The value was calculated using the following formula. I C 50 (nM) = antilog(LOG 10 (a÷b)×(ed)÷(cd)+LOG 10 b) a: concentration of test substance a b: concentration of test substance b c: Cell viability when test substance at concentration a was added d: Cell viability when test substance at concentration b was added e: The mean value between the maximum and minimum cell viability when each concentration of the test substance was added. (a and b are concentrations that straddle the cell viability e, and a>b represents the concentration.)

[0473] The cell viability at each concentration was calculated using the following formula. Cell viability (%)=a'÷b'×100 a': Average luminescence intensity of wells to which the test substance was added (n=6) b': Average luminescence intensity in wells to which no test substance was added (n=6) (n represents the number of evaluations performed per concentration of the test substance.)

[0474] [Table 24-1] [Table 24-2]

[0475] As shown in Table 24 above, the comparative compound exhibited strong cytotoxicity against Karpas-299 cells. On the other hand, the example compound exhibited weak cytotoxicity against Karpas-299 cells. This difference in cytotoxicity is presumed to be due to the difference in membrane permeability shown in Test Example 4.

[0476] Test Example 2: Cytotoxicity test (2) SK-BR-3 cells (ATCC), a human breast cancer cell line, were cultured in McCoy's 5A (GIBCO) containing 10% fetal bovine serum (MP Biomedicals) (hereinafter referred to as "medium" in this study). SK-BR-3 cells were cultured at 2 × 10 6 The cells / mL were adjusted, and 50 μL of each was added to a 96-well cell culture microplate. After overnight incubation at 37°C and 5% CO2, 50 μL of the compound of the Example or the Comparative Example compound, which had been diluted 4-fold in 8 steps with the medium, was added to the microplate. These were cultured at 37°C and 5% CO2 for 3 days. After incubation, the microplate was removed from the incubator and left to stand at room temperature for 10 minutes. 50 μL of CellTiter-Glo Luminescent Cell Viability Assay (Promega) was added to each well and stirred. This mixture was incubated in the dark for 20 minutes. The cell viability was calculated by measuring the luminescence in each well using a microplate luminometer. IC 50 The values ​​were calculated according to the method described in Test Example 1. The results are shown in Table 25.

[0477] [Table 25]

[0478] As shown in Table 25 above, the comparative compound exhibited strong cytotoxicity against SK-BR-3 cells. On the other hand, the example compound exhibited weak cytotoxicity against SK-BR-3 cells. This difference in cytotoxicity is presumed to be due to the difference in membrane permeability shown in Test Example 4.

[0479] Test Example 3: Cytotoxicity test of ADC (1) Karpas-299 cells (ECACC), which are CD30 antigen positive and HER2 antigen negative, were cultured in RPMI1640 (GIBCO) containing 10% fetal bovine serum (MP Biomedicals) (hereinafter referred to as "medium A" in this test). SK-BR-3 cells (ATCC), which are CD30 antigen negative and HER2 antigen positive, were cultured in McCoy's 5A (GIBCO) containing 10% fetal bovine serum (MP Biomedicals) (hereinafter referred to as "medium B" in this test). Karpas-299 cells and SK-BR-3 cells were cultured in medium A or medium B at a concentration of 2 × 10 6 The ADC was adjusted to 100 μL / mL and added to a 96-well cell culture microplate at 50 μL each. After addition, SK-BR-3 cells were cultured overnight at 37°C and 5% CO2. 50 μL each of ADC diluted 4-fold in 8 steps with medium A or medium B was added to the microplate, and Karpas-299 cells were cultured for 4 days and SK-BR-3 cells were cultured for 3 days at 37°C and 5% CO2. After culture, the microplate was removed from the incubator and left to stand at room temperature for 10 minutes. 50 μL of CellTiter-Glo Luminescent Cell Viability Assay (Promega) was added to each well and stirred. This mixture was incubated in the dark for 20 minutes. The cell viability was calculated for each ADC concentration by measuring luminescence using a microplate luminometer. IC 50 The values ​​were calculated according to the method described in Test Example 1. The results are shown in Table 26.

[0480] [Table 26-1] [Table 26-2]

[0481] As shown in Table 26 above, Example ADC1 (an antibody-drug conjugate of brentuximab, a CD30 antigen-specific antibody, and Example M3) showed strong cytotoxic activity against Karpas-299 cells, which are CD30 antigen-positive cells, but showed weak cytotoxic activity against SK-BR-3 cells, which are CD30 antigen-negative cells. Similarly, Example ADC4 (an antibody-drug conjugate of brentuximab, a CD30 antigen-specific antibody, and Example M4) also showed strong cytotoxic activity against Karpas-299 cells, which are CD30 antigen-positive cells, and showed weak cytotoxic activity against SK-BR-3 cells, which are CD30 antigen-negative cells. In addition, Example ADC9 (an antibody-drug conjugate of trastuzumab, a HER2 antigen-specific antibody, and Example M3) showed strong cytotoxic activity against SK-BR-3 cells, which are HER2 antigen-positive cells, and showed weak cytotoxic activity against Karpas-299 cells, which are HER2 antigen-negative cells.

[0482] In other words, even a compound with weak cytotoxicity due to low membrane permeability (corresponding to the compound in the Examples) showed selectively strong cytotoxic activity against antigen-positive cells that specifically bind to the antibody by forming a complex with the antibody (corresponding to the ADC in the Examples).

[0483] Test Example 4: Membrane permeability test The membrane permeability of the compounds of the examples was tested by the artificial membrane permeability test (PAMPA) as follows. 200 μL of system solution (pION inc.) containing the compound of the examples and 4 μL of GIT Lipid-0 (pION inc.) were added to the donor plate. 200 μL of Acceptor Sink Buffer (pION inc.) was added to the acceptor plate. Both plates were overlaid and incubated at 37° C. for 4 hours, after which the UV of the solutions on the acceptor side and the donor side was measured using a UV plate reader (190-500 nm). Compounds with poor UV absorption were measured using LC-MS. The permeability coefficient P of the druge (10 -6 The velocity (cm / sec) was calculated using the following formula. The results are shown in Table 27.

number

[0484] [Table 27-1] [Table 27-2]

[0485] From the results of Test Examples 1 to 4, it is presumed that the compounds of the Examples showed lower activity than the compounds of the Comparative Examples in the cytotoxicity test due to the difference in cell membrane permeability between these compounds. That is, it is considered that the compounds of the Examples showed weaker cytotoxicity than the compounds of the Comparative Examples because the compounds of the Examples had low cell membrane permeability and the migration of the compounds of the Examples into cells was suppressed. Therefore, it is suggested that the antibody-drug conjugates containing the compounds of the Examples can suppress the migration of the compounds of the Examples, which have low cell membrane permeability, into normal cells even if reversible dissociation between the antibody moiety and the drug moiety occurs in the systemic blood, thereby reducing side effects.

[0486] Test Example 5: Efficacy test of antibody-drug conjugates in Karpas-299 tumor type using CB-17 SCID mice This test is a representative test for evaluating the antitumor activity of drugs. The Karpas human anaplastic large cell lymphoma model is performed in CB-17 SCID mice at 5 × 10 6 The tumor model is created by subcutaneously transplanting 100 cells. 3 Treatment was initiated after the mean volume was reached. Mice were given a single intravenous injection of ADC in phosphate buffered saline. Tumor volume was calculated using the formula: 0.5(longest dimension x perpendicular dimension) 2 ) is used to calculate the tumor size. 3When the tumor size reaches 100%, the mouse is removed from the study and the average tumor size is no longer plotted. The method of this study is described in Hamblett KJ et. al. Clin. Cancer Res., 2004, 10, 7063-7070, etc.

[0487] Test Example 6: Toxicity (safety) test of drugs or antibody-drug conjugates using mice or rats This test is a typical test to evaluate the toxicity (safety) of antibody-drug conjugates. Toxicity can be confirmed by administering the antibody-drug conjugate once or repeatedly into the tail vein of mice or rats, and then observing general symptoms, hematological tests, blood biochemistry tests, bone marrow tests, autopsy, organ weights, histopathological tests, etc. This test is described in New Edition Toxicology, edited by the Education Committee of the Japanese Society of Toxicology, Asakura Publishing (2009), and in the Brentuximab Vetotin Application Document Summary, Pharmaceuticals and Medical Devices Agency, etc.

[0488] Test Example 7: Cytotoxicity test of ADC (2) Cytotoxicity was measured using Examples ADC17, ADC18, ADC41, and ADC42. Specifically, Karpas-299 cells (ECACC) were cultured in RPMI1640 (GIBCO) containing 10% fetal bovine serum (MP Biomedicals) (hereinafter referred to as "medium A" in this test). Karpas-299 cells were cultured in medium A at a concentration of 2 × 10 6The cells / mL were adjusted and 50 μL was added to a 96-well cell culture microplate. Brentuximab or the example ADC, adjusted to a concentration of 2000 ng / mL in medium A, was added to the microplate in 50 μL portions and cultured at 37 degrees and 5% CO2 for 4 days. After culture, the microplate was removed from the incubator and left to stand at room temperature for 10 minutes. 50 μL of CellTiter-Glo Luminescent Cell Viability Assay (Promega) was added to each well and stirred. This mixture was incubated in the dark for 20 minutes, and the luminescence was measured using a microplate luminometer to determine the percentage of the number of cells remaining when treated with the example ADC relative to the number of cells remaining when treated with brentuximab. The results are shown in the table below. [Table 28]

[0489] As shown in Test Example 7, when the ADC of the Example was used, a more significant decrease in cell count was observed compared to when its antibody moiety, brentuximab, was used, demonstrating that the ADC of the present invention exhibits stronger cytotoxicity than the antibody itself.

[0490] Test Example 8: Cytotoxicity test of ADC (3) The cytotoxicity of the ADCs of the examples was measured using the cell types shown in the table below according to the method described in Test Example 3 or Test Example 7. The IC 50 The survival rate of cells was measured when treated with 333 nM ADC or 333 nM ADC. However, in the case of ADC51, the survival rate of cells was measured when treated with 26.6 nM ADC51. The results are shown in the table below. [Table 29]

[0491] As shown in the table above, the example ADCs exhibited cytotoxic activity against cells expressing the antigen to which the antibody portion of the ADC binds.

[0492] From the results of Test Examples 1 to 8, it was found that the compounds of the Examples showed lower activity than the compounds of the Comparative Examples in the cytotoxicity test. On the other hand, the antibody-drug conjugate obtained by binding the compounds of the Examples to the antibody showed high activity in the cytotoxicity test. From these results, it is presumed that the difference in cytotoxicity between the compounds of the Comparative Examples and the compounds of the Examples is based on the difference in cell membrane permeability. That is, it is considered that the compounds of the Examples showed weaker cytotoxicity than the compounds of the Comparative Examples because the compounds of the Examples had low cell membrane permeability and the migration of the compounds of the Examples into cells was suppressed. Therefore, it was suggested that the antibody-drug conjugates containing the compounds of the Examples could suppress the migration of the compounds of the Examples, which have low cell membrane permeability, into normal cells even if reversible dissociation between the antibody portion and the drug portion occurs in the systemic blood, thereby reducing side effects. [Industrial Applicability]

[0493] As explained above, the antibody-drug conjugate of the present invention selectively exhibits cytotoxic activity in antigen-expressing cells and has low cytotoxicity in normal cells, and is therefore expected to become an anticancer drug with excellent safety.

Claims

1. Formula (1-1): 【Chemistry 1】 [In the formula, b represents an integer of 1 to 4; Z is represented by formula (Z-1), formula (Z-2) or formula (Z-3): 【Chemistry 2】 (In the formula, n represents an integer of 0 to 2; AA represents a glutamic acid residue (Glu) or an aspartic acid residue (Asp). When there are a plurality of AAs, each AAs may be the same or different from each other, and the AAs are bonded to each other via an amide bond. (AA) ｎ The N-terminal nitrogen atom of the formula (I) forms an amide bond with the carbonyl group (a), Q is represented by formula (Qa-1) or formula (Qa-2): 【Chemistry 3】 (In the formula, R ａｂ and R ａｃ each independently represents a hydrogen atom or a halogen atom, R ａｄ , R ａｅ and R ａｆ each independently represents a hydrogen atom, a fluorine atom, a carboxyl group, a phenyl group, or a C optionally substituted by 1 to 3 fluorine atoms １－６ Alkyl group or C １－６ represents an alkoxy group) represents a group represented by R １ａ and R １ｂ each independently represents a hydrogen atom or a methyl group; R ２ is -(CH ２ ) ｕ -COR ４ represents u represents 1 or 2; R3 is -OH or -(AB) ｐ represents AB represents Glu or Asp; p represents 1; R 4 represents —OH. is a group represented by the formula: A compound or a salt thereof represented by the following formula: (However, compounds selected from the following or salts thereof are excluded.) 【Chemistry 4】

2. Formula (1-1): 【Chemistry 5】 [In the formula, b represents an integer of 2 to 4; Z is represented by formula (Z-1), formula (Z-2) or formula (Z-3): 【Chemistry 6】 (In the formula, n represents an integer of 0 to 2; AA represents a glutamic acid residue (Glu) or an aspartic acid residue (Asp). When there are a plurality of AAs, each AAs may be the same or different from each other, and the AAs are bonded to each other via an amide bond. (AA) ｎ The N-terminal nitrogen atom of the formula (I) forms an amide bond with the carbonyl group (a), Q is an unsubstituted phenyl group or a group represented by formula (Q-1): 【Chemistry 7】 represents a group represented by R １ａ and R １ｂ each independently represents a hydrogen atom or a methyl group; R ２ is -(CH ２ ) ｕ -COR ４ represents u represents 1 or 2; R3 is -OH or -(AB) ｐ represents AB represents Glu or Asp; p represents 1; R 4 represents —OH. is a group represented by the formula: The compound according to claim 1 or a salt thereof,

3. 3. The compound or salt thereof according to claim 1 or 2, wherein b is 2.

4. The compound or salt thereof according to any one of claims 1 to 3, wherein n is 0.

5. R3 is -(AB) ｐ and the sum of n and p is 1 or 2, or a salt thereof according to any one of claims 1 to 3.

6. R ３ and R ４ is —OH, The compound or salt thereof according to any one of claims 1 to 3, wherein n is 0.

7. The compound or salt thereof according to any one of claims 1 to 3, wherein AA represents a glutamic acid residue (Glu), and when there are a plurality of AA, the AA are bonded to each other via an amide bond.

8. Formula (1-2): 【Chemistry 8】 [In the formula, h represents an integer of 3 to 5; Z′ is represented by formula (Z-4) or formula (Z-5): 【Chemistry 9】 (In the formula, n represents 0 or 1; AA represents a glutamic acid residue (Glu) or an aspartic acid residue (Asp); (AA) ｎ The N-terminal nitrogen atom of the formula (I) forms an amide bond with the carbonyl group (a), Q is an unsubstituted phenyl group or a group represented by formula (Q-1): 【Chemistry 10】 represents a group represented by R １ａ and R １ｂ each independently represents a hydrogen atom or a methyl group. is a group represented by the formula: A compound or a salt thereof represented by the following formula: (However, the compounds or salts thereof described below are excluded.) 【Chemistry 11】

9. The compound or salt thereof according to claim 8, wherein h is 5.

10. 10. The compound or salt thereof according to claim 8 or 9, wherein n is 0.

11. Q is a group represented by formula (Qa-1) or formula (Qa-2), R ａｂ and R ａｃ are each independently a hydrogen atom or a fluorine atom, R ａｄ , R ａｅ and R ａｆ The compound or salt thereof according to claim 1 , wherein each of the is independently a hydrogen atom or a fluorine atom.

12. Formula (2-1): 【Chemistry 12】 [In the formula, mAb stands for antibody; q represents an integer of 1 to 8; b represents an integer of 1 to 4; Z is represented by formula (Z-1), formula (Z-2) or formula (Z-3): 【Chemistry 13】 (In the formula, n represents an integer of 0 to 2; AA represents a glutamic acid residue (Glu) or an aspartic acid residue (Asp). When there are a plurality of AAs, each AAs may be the same or different from each other, and the AAs are bonded to each other via an amide bond. (AA) ｎ The N-terminal nitrogen atom of the formula (I) forms an amide bond with the carbonyl group (a), Q is represented by formula (Qa-1) or formula (Qa-2): 【Chemistry 14】 (In the formula, R ａｂ and R ａｃ each independently represents a hydrogen atom or a halogen atom, R ａｄ , R ａｅ and R ａｆ each independently represents a hydrogen atom, a fluorine atom, a carboxyl group, a phenyl group, or a C optionally substituted by 1 to 3 fluorine atoms １－６ Alkyl group or C １－６ represents an alkoxy group) represents a group represented by R １ａ and R １ｂ each independently represents a hydrogen atom or a methyl group; R ２ is -(CH ２ ) ｕ -COR ４ represents u represents 1 or 2; R3 is -OH or -(AB) ｐ represents AB represents Glu or Asp; p represents 1; R 4 represents —OH. is a group represented by the formula: An antibody-drug conjugate or a pharma- ceutical acceptable salt thereof, (However, antibody-drug conjugates selected from the following or pharma- ceutically acceptable salts thereof are excluded.) 【Chemistry 15】 [In the formula, mAb represents an antibody, and q represents an integer from 1 to 8.]

13. Formula (2-1): 【Chemistry 16】 [In the formula, mAb stands for antibody; q represents an integer of 1 to 8; b represents an integer of 2 to 4; Z is represented by formula (Z-1), formula (Z-2) or formula (Z-3): 【Chemistry 17】 (In the formula, n represents an integer of 0 to 2; AA represents a glutamic acid residue (Glu) or an aspartic acid residue (Asp). When there are a plurality of AAs, each AAs may be the same or different from each other, and the AAs are bonded to each other via an amide bond. (AA) ｎ The N-terminal nitrogen atom of the formula (I) forms an amide bond with the carbonyl group (a), Q is an unsubstituted phenyl group or a group represented by formula (Q-1): 【Chemistry 18】 represents a group represented by R １ａ and R １ｂ each independently represents a hydrogen atom or a methyl group; R ２ is -(CH ２ ) ｕ -COR ４ represents u represents 1 or 2; R3 is -OH or -(AB) ｐ represents AB represents Glu or Asp; p represents 1; R 4 represents —OH. is a group represented by the formula: The antibody-drug conjugate or a pharma- ceutically acceptable salt thereof according to claim 12, wherein

14. The antibody-drug conjugate or a pharma- ceutically acceptable salt thereof according to claim 12 or 13, wherein b is 2.

15. The antibody-drug conjugate or a pharma- ceutically acceptable salt thereof according to any one of claims 12 to 14, wherein n is 0.

16. R3 is -(AB) ｐ and the sum of n and p is 1 or 2. The antibody-drug conjugate or a pharma- ceutically acceptable salt thereof according to any one of claims 12 to 14.

17. R ３ and R ４ is —OH, The antibody-drug conjugate or a pharma- ceutically acceptable salt thereof according to any one of claims 12 to 14, wherein n is 0.

18. b is 2; Z is a group represented by formula (Z-3), n is 0, Q is an unsubstituted phenyl group; R １ａ is a methyl group, R １ｂ is a hydrogen atom, R ３ The antibody-drug conjugate or a pharma- ceutically acceptable salt thereof according to any one of claims 12 to 14, wherein is -OH.

19. Formula (2-2): 【Chemistry 19】 [In the formula, mAb stands for antibody; q represents an integer of 1 to 8; h represents an integer of 3 to 5; Z′ is represented by formula (Z-4) or formula (Z-5): 【Chemistry 20】 (In the formula, n represents 0 or 1; AA represents a glutamic acid residue (Glu) or an aspartic acid residue (Asp); (AA) ｎ The N-terminal nitrogen atom of the formula (I) forms an amide bond with the carbonyl group (a), Q is an unsubstituted phenyl group or a group represented by formula (Q-1): 【Chemistry 21】 represents a group represented by R １ａ and R １ｂ each independently represents a hydrogen atom or a methyl group. is a group represented by the formula: An antibody-drug conjugate or a pharma- ceutically acceptable salt thereof,

20. 20. The antibody-drug conjugate or a pharma- ceutically acceptable salt thereof of claim 19, wherein h is 5.

21. 21. The antibody-drug conjugate or a pharma- ceutically acceptable salt thereof according to claim 19 or 20, wherein n is 0.

22. Q is a group represented by formula (Qa-1) or formula (Qa-2), R ａｂ and R ａｃ are each independently a hydrogen atom or a fluorine atom, R ａｄ , R ａｅ and R ａｆ The antibody-drug conjugate or a pharma- ceutically acceptable salt thereof according to claim 12, wherein each of the is independently a hydrogen atom or a fluorine atom.

23. The mAbs are brentuximab, trastuzumab, inotuzumab, gemtuzumab, glembatumumab, labetuzumab, sacituzumab, rifastuzumab, indusatuzumab, polatuzumab, pinatuzumab, cortuximab, indatuximab, milatuzumab, rovalpituzumab, anetumab, tisotumab, miabetuximab, lorvotuzumab, rituximab, depatuxizumab, denintuzumab, enfortumab, tersotuzumab, bundletuzumab, sofituzumab, borsetuzumab, mirvetuximab, naratuximab, cantuzumab, and laprituximab. , bivatuzumab, vadastuximab, rupartumab, apurutumab, abagovomab, abciximab, avituzumab, abrilumab, actoxumab, adalibumab, adecatumumab, aducanumab, afacevicumab, afelimomab, alacizumab, alemtuzumab, alirocumab, altumomab, amatuximab, anatumomab, anifrolumab, anrukinzumab, apolizumab, arcitumomab, asclinbacumab, acelizumab, atezolizumab, atinumab, atorlimumab, avelumab, azintuxizumab, bapineuzumab, basilicumab Cimab, bavituximab, bectumomab, begelomab, belimumab, benralizumab, bertilimumab, besilesomab, bevacizumab, bezlotoxumab, biciromab, bimagrumab, bimekizumab, bleselumab, blinatumomab, brontuzumab, brosozumab, bococizumab, brazikumab, briakinumab, brodalumab, brolucizumab, broticutuzumab, burosumab, cabilalizumab, camrelizumab, caplacizumab, capromab, carlumab, carotuximab, catumazomab, cedelizumab, certolizumab, cetuximab, sitat Zumab, sikisumumab, serenoliximab, clivatuzumab, codrituzumab, conatumumab, concizumab, cosfrobiximab, crenezumab, crizanlizumab, cloteduumab, dacetuzumab, daclizumab, dalotuzumab, dapirorizumab, daratumumab, dectrekumab, demcizumab, denosumab, detumomab, dezamizumab, dinutuximab, zilidamab, domagurozumab, dorlimomab, drozitumab, durigotuzumab, dupilumab, durvalumab, dusigitumab, duvortuxizumab, ecromeximab, eculizumab,Edobacomab, edrecolomab, efalizumab, efungumab, eldelumab, elezamumab, elotuzumab, ersilimomab, emactuzumab, emapalumab, emibetuzumab, emicizumab, enavatuzumab, enlimomab, enoblitzumab, enokizumab, enoticumab, encituximab, epitumomab, epratuzumab, eptinezumab, erenumab, erlizumab, ertumaxomab, etaracizumab, etrolizumab, evinacumab, evolocumab, exbivirumab, faralimomab, farletuzumab, fasinumab, felvisumab , fezakinumab, ficlatuzumab, figitumumab, filibumab, flavotumab, fretikumab, fontolizumab, foralumab, foravirumab, fremanezumab, fresolimumab, fulnevetomab, fullanumab, futuximab, galcanezumab, galiximab, ganitumab, gantenerumab, gatipotuzumab, gavilimomab, gedivumab, gevokizumab, gilvetumab, girentuximab, golimumab, guselkumab, ibalizumab, ibritumomab, icrucumab, idarucizumab, ifavotuzumab, igovomab, imalumab, im Ciromab, Imgatuzumab, Inclacumab, Inebilizumab, Infliximab, Inolimomab, Intetumumab, Ipilimumab, Iratumumab, Isatuximab, Itolizumab, Ixekizumab, Keliximab, Lacnotumab, Lampalizumab, Lanadelumab, Landgrozumab, Ralcaviximab, Lebrikizumab, Remaresomab, Lenzilumab, Lerdelimumab, Lesofabutumab, Letolizumab, Lexatumumab, Ribililumab, Rifatuzumab, Ligelizumab, Rilotomab, Lintuzumab, Lirilumab, Rodericizumab, Loxobetomab, Rovo ... Zumab, rosatuximab, lucatumumab, lurizumab, lumuletuzumab, rutikizumab, mapatumumab, margetuximab, masalimoab, matuzumab, mavrilimumab, mepolizumab, metelimumab, minitelumomab, mitumomab, modotuximab, mogamulizumab, monalizumab, morolizumab, motavizumab, moxetumomab, muromonab, nacolomab, namilumab, naptumomab, narnalumab, natalizumab, navicixizumab, nabivumab, nebacumab, necitumumab, nemolizumab, nerelimomab, nesbacumab, nimotuzumab, nivolumab,Oviltoxaximab, obinutuzumab, ocaratuzumab, ocrelizumab, odulimomab, ofatumumab, olaratumab, oleculumab, orendalizumab, olokizumab, omalizumab, onartuzumab, ontuxizumab, opicinumab, oportuzumab, oregovomab, oreticumab, olticumab, otelixizumab, otlertuzumab, oxelumab, ozanezumab, ozoralizumab, pagibaximab, palivizumab, pamrevlumab, panitumumab, panobakumab, palsatuzumab, pascolizumab, pasotuxizumab, patekrisumab , patritumab, pembrolizumab, perakizumab, pertuzumab, pexelizumab, pidilizumab, placumab, prozalizumab, ponezumab, polgabiximab, prezalumab, priliximab, plitoxaximab, pritumumab, quilizumab, racotumomab, radletumab, rafivirumab, ralpancizumab, ramucirumab, ranevetomab, ranibizumab, raxibacumab, refanezumab, regavirumab, lemtolumab, reslizumab, rilotumumab, linikumab, risankizumab, rivavazumab, lobatumumab, romot ... Sozumab, Rontalizumab, Rosumantuzumab, Rovelizumab, Rozanolixizumab, Ruplizumab, Samalizumab, Sarilumab, Satralizumab, Satumomab, Secukinumab, Celicrelumab, Seribantumab, Setoxaximab, Sevirumab, Sibrotuzumab, Sifarlimumab, Siltuximab, Simtuzumab, Siplizumab, Sirukumab, Solanezumab, Solitomab, Sontuzumab, Stamulumab, Sulesomab, Sumtabumab, Suvisumab, Subratocumab, Tabalumab, Tadocizumab, Talizumab, Tamtubetomab, Tanezumab, Taplitumomab , talexuzumab, tabolixizumab, fanolesomab, nofetumomab, pintumomab, tefibazumab, terimomab, telisozumab, tenatumomab, teneliximab, teplizumab, teprotumumab, tesidolumab, tezepelumab, tigatuzumab, tildrakizumab, timigtuzumab, timolumab, tocilizumab, tomzotuximab, toralizumab, tosatoxumab, tositumomab, tobetumab, tralokinumab, tregalizumab, tremelimumab, trevoglumab, tucotuzumab, tuvilumab, ublituximab, urocuprumab, urelumab,The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 12 to 22, which is an antibody selected from the group consisting of urtoxazumab, ustekinumab, utomilumab, vanticutumab, vanucizumab, bapaliximab, valisacumab, varlilumab, batelizumab, vedolizumab, beltuzumab, beparimomab, besencumab, visilizumab, bovalilizumab, volociximab, bonlerolizumab, votumumab, bunakizumab, tacatuzumab, zaltuzumab, zanolimumab, diralimumab, zolimomab, camidanlumab, cofetuzumab, radilatuzumab, loncastuzumab, terisotuzumab, enapotamab, and AMG595, or an anti-emvidin antibody.

24. 24. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof of claim 23, wherein the mAb is brentuximab, trastuzumab, inotuzumab, gemtuzumab, labetuzumab, polatuzumab, cortuximab, indatuximab, anetuzumab, rituximab, denintuzumab, laprituximab, vadastuximab, glembatumumab, cetuximab, alemtuzumab, or depatuxizumab.

25. 25. The antibody-drug conjugate or a pharma- ceutically acceptable salt thereof of claim 24, wherein the mAb is brentuximab or trastuzumab.

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